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RAF1
Available structures
PDBOrtholog search: PDBe RCSB
Identifiers
AliasesRAF1, Raf-1 proto-oncogene, serine/threonine kinase, CMD1NN, CRAF, NS5, Raf-1, c-Raf
External IDsOMIM: 164760; MGI: 97847; HomoloGene: 48145; GeneCards: RAF1; OMA:RAF1 - orthologs
Orthologs
SpeciesHumanMouse
Entrez

5894

110157

Ensembl

ENSG00000132155

ENSMUSG00000000441

UniProt

P04049

Q99N57

RefSeq (mRNA)

NM_002880

NM_029780
NM_001356333
NM_001356334

RefSeq (protein)

NP_084056
NP_001343262
NP_001343263

Location (UCSC)n/aChr 6: 115.6 – 115.65 Mb
PubMed search[2][3]
Wikidata
View/Edit HumanView/Edit Mouse

RAF proto-oncogene serine/threonine-protein kinase, also known as proto-oncogene c-RAF or simply c-Raf or even Raf-1, is an enzyme[4] that in humans is encoded by the RAF1 gene.[5][6] The c-Raf protein is part of the ERK1/2 pathway as a MAP kinase (MAP3K) that functions downstream of the Ras subfamily of membrane associated GTPases.[7] C-Raf is a member of the Raf kinase family of serine/threonine-specific protein kinases, from the TKL (Tyrosine-kinase-like) group of kinases.

Discovery

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The first Raf gene, v-Raf was found in 1983. It was isolated from the murine retrovirus bearing the number 3611. It was soon demonstrated to be capable to transform rodent fibroblasts to cancerous cell lines, so this gene was given the name Virus-induced Rapidly Accelerated Fibrosarcoma (V-RAF).[5] A year later, another transforming gene was found in the avian retrovirus MH2, named v-Mil - that turned out to be highly similar to v-Raf.[8] Researchers were able to demonstrate that these genes encode enzymes that have serine-threonine kinase activity.[9] Normal cellular homologs of v-Raf and v-Mil were soon found in both the mouse and chicken genome (hence the name c-Raf for the normal cellular Raf gene), and it became clear that these too had a role in regulating growth and cell division.[10][11]

c-Raf is a principal component of the mitogen-activated protein kinase (MAPK) pathway: ERK1/2 signaling.[12] It acts as a MAP3 kinase, initiating the entire kinase cascade. Subsequent experiments showed that the normal, cellular Raf genes can also mutate to become oncogenes, by "overdriving" MEK1/2 and ERK1/2 activity.[13] In fact, vertebrate genomes contain multiple Raf genes. Several years later after the discovery of c-Raf, two further related kinases were described: A-Raf and B-Raf. The latter became the focus of research in recent years, since a large portion of human tumors carry oncogenic 'driver' mutations in the B-Raf gene.[14] These mutations induce an uncontrolled, high activity of Raf enzymes. Thus diagnostic and therapeutic interest in Raf kinases reached a new peak in the recent years.[15]

Structure

[edit]

The human c-Raf gene is located on chromosome 3. At least two isoforms of mRNA have been described (arising from inclusion or removal of an alternative exon) that display only minute differences. The shorter, major isoform - consisting of 17 exons - encodes a protein kinase of 648 amino acids.[16]

A schematic architecture of human c-Raf protein

Similarly to many other MAPKKKs, c-Raf is a multidomain protein, with several additional domains to aid the regulation of its catalytic activity. On its N-terminal segment, a Ras-binding domain (RBD) and a C-kinase homologous domain 1 (C1 domain) are found next to each other. Structures of both conserved domains were solved in the past decades, shedding light on the mechanisms of their regulation.

The Ras-binding domain displays a ubiquitin-like fold (like many other small G-protein associating domains) and selectively binds GTP-bound Ras proteins only.[17][18][19] (You can see this interaction in high detail in the PDB box attached to the article. It shows Rap1 in complex with the RBD of c-Raf.)

The C1 domain - immediately downstream of the Ras binding domain - is a special zinc finger, rich in cysteines and stabilized by two zinc ions. It is similar to the diacylglycerol-binding C1 domains of protein kinase C (PKC) enzymes.[20][21] But unlike PKC, the C1 domains of Raf family kinases do not bind diacylglycerol.[22] Instead, they interact with other lipids, such as ceramide[22] or phosphatidic acid,[23] and even aid in the recognition of activated Ras (GTP-Ras).[21][24]

The close proximity of these two domains as well as several lines of experimental data suggest that they act as a single unit to negatively regulate the activity of the protein kinase domain, by direct physical interaction.[25] Historically, this autoinhibitory block was labelled as the CR1 region ("Conserved Region 1"), the hinge region being named CR2, and the kinase domain CR3. Unfortunately, the precise structure of the autoinhibited kinase remains unknown.

Between the autoinhibitory domain block and the catalytic kinase domain, a long segment - characteristic to all Raf proteins - can be found. It is highly enriched in serine amino acids, but its precise sequence is poorly conserved across related Raf genes. This region appears to be intrinsically unstructured, and very flexible. Its most likely role is to act as a natural "hinge" between the rigidly folded autoinhibitory and catalytic domains, enabling complex movements and profound conformational rearrangements within the molecule.[26] This hinge region contains a small, conserved island of amino acids, that are responsible for 14-3-3 protein recognition, but only when a critical serine (Ser259 in human c-Raf) is phosphorylated. A second, similar motif is found on the extreme C-terminus (centered around the phosphorylatable Ser 621) of all Raf enzymes, but downstream of the kinase domain.

The C-terminal half of c-Raf folds into a single protein domain, responsible for catalytic activity. The structure of this kinase domain is well-known from both c-Raf[27] and B-Raf.[28] It is highly similar to other Raf kinases and KSR proteins, and distinctly similar to some other MAP3 kinases, such as the Mixed Lineage Kinase (MLK) family. Together they comprise the Tyrosine Kinase Like (TKL) group of protein kinases. Although some features unite their catalytic domains with protein tyrosine kinases, the activity of TKLs is restricted to the phosphorylation of serine and threonine residues within target proteins. The most important substrate of Raf kinases (apart from itself) are the MKK1 and MKK2 kinases, whose activity strictly depends on phosphorylation events performed by Rafs.

Evolutionary relationships

[edit]
Main article: Raf kinase

Human c-Raf is a member of a larger family of related protein kinases. Two further members - found in most vertebrates - belong to the same family: B-Raf and A-Raf. Apart from the different length of their non-conserved N- and C-terminal ends, they all share the same domain architecture, structure and regulation. In comparison to the relatively well-known c-Raf and B-Raf, there is very little known of the precise function of A-Raf, but it is also thought to be similar to the other two members of the family. All these genes are believed to be the product of full gene or genome duplications at the dawn of vertebrate evolution, from a single ancestral Raf gene. Most other animal organisms possess only a single Raf gene. It is called Phl or Draf in Drosophila[29] and Lin-45 in C. elegans.[30]

The family of Raf kinases (schematic architectures)

Multicellular animals also have a type of kinase closely related to Raf: this is the Kinase Suppressor of Ras (KSR). Vertebrates like mammals have two, paralogous KSR genes instead of one: KSR1 and KSR2. Their C-terminal kinase domain is very similar to Raf (originally called CA5 in KSR and CR3 in Raf), but the N-terminal regulatory region differs. Although they also have the flexible hinge (CA4 in KSR) and a C1 domain (CA3 in KSR) before it, KSRs entirely lack the Ras-binding domain. Instead, they have unique regulatory regions on their N-termini, originally termed CA1 ("conserved area 1") and CA2. For a long time, the structure of the CA1 domain was a mystery. However, in 2012, the structure of the CA1 region in KSR1 was solved: it turned out to be a divergent SAM (sterile alpha motif) domain, supplemented with coiled-coils (CC-SAM): this is supposed to aid KSRs in membrane binding.[31] KSRs, like Rafs, also have the twin 14-3-3 associating motifs (that depend on phosphorylation), but also possess novel MAPK-binding motifs on their hinge regions. With a typical sequence Phe-x-Phe-Pro (FxFP) these motifs are important for the feedback regulation of Raf kinases in the ERK1/2 pathway. According to our current knowledge, KSRs also participate in the same pathway as Raf, although they only play an auxiliary role. With a very poor intrinsic kinase activity, they were long thought to be inactive, until their catalytic activity was finally demonstrated in recent years.[32][33] But even then, they contribute only negligibly to MKK1 and MKK2 phosphorylation. The main role of KSR appears to be to provide a heterodimerization partner to Raf enzymes, greatly facilitating their activation by means of allostery. Similar phenomena were described for other MAP3 kinases. ASK2, for example, is a poor enzyme on its own, and it's activity appears to be tied to ASK1/ASK2 heterodimerisation.[34]

Raf-like kinases are fully absent from fungi. But recent sequencing of other opisthokonts (e.g. Capsaspora owczarzaki) revealed the presence of genuine Raf kinases in unicellular eukaryotes. Therefore, it is possible that Raf proteins are an ancient heritage and ancestors of fungi secondarily lost Raf-dependent signaling. Fungal MAP kinase pathways that are homologous to the mammalian ERK1/2 pathway (Fus3 and Kss1 in yeast) are activated by MEKK-related kinases (e.g. Ste11 in yeast) instead of Raf enzymes.

Raf kinases found in retroviruses (such as murine v-Raf) are secondarily derived from the corresponding vertebrate genes of their hosts. These Raf genes encode severely truncated proteins, that lack the entire N-terminal autoinhibitory domain, and the 14-3-3 binding motifs. Such severe truncations are known to induce an uncontrolled activity of Raf kinases: that is just exactly what a virus may need for efficient reproduction.

Regulation of activity

[edit]
Artist's impression of the autoinhibited state of c-Raf, reinforced by the associated 14-3-3 protein dimers, bound to the phosphorylated twin motifs.[35][36]

As mentioned above, the regulation of c-Raf activity is complex. As a "gatekeeper" of the ERK1/2 pathway, it is kept in check by a multitude of inhibitory mechanisms, and normally cannot be activated in a single step. The most important regulatory mechanism involves the direct, physical association of the N-terminal autoinhibitory block to the kinase domain of c-Raf. It results in the occlusion of the catalytic site and full shutdown of kinase activity.[25] This "closed" state can only be relieved if the autoinhibitory domain of Raf engages a partner competing with its own kinase domain, most importantly GTP-bound Ras. Activated small G-proteins can thus break up the intramolecular interactions: this results in a conformational change ("opening") of c-Raf[37] necessary for kinase activation and substrate binding.

14-3-3 proteins also contribute to the autoinhibition. As 14-3-3 proteins are all known to form constitutive dimers, their assemblies have two binding sites.[38] Thus the dimer acts as a "molecular handcuff", locking their binding partners at a fixed distance and orientation. When the precisely positioned twin 14-3-3 binding motifs are engaged by a single 14-3-3 protein dimer (such as 14-3-3 zeta), they become locked into a conformation that promotes autoinhibition and does not allow the disengagement of the autoinhibitory and catalytic domains.[39] This "lockdown" of c-Raf (and other Rafs as well as KSRs) is controlled by motif phosphorylation. Unphosphorylated 14-3-3 associating motifs do not bind their partners: they need to get phosphorylated on conserved serines (Ser 259 and Ser 621) first, by other protein kinases. The most important kinase implicated in this event is TGF-beta activated kinase 1 (TAK1), and the enzymes dedicated for removal of these phosphates are the protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) complexes.[40][41]

Note that 14-3-3 binding of Raf enzymes is not necessarily inhibitory: once Raf is open and dimerizes, 14-3-3s can also bind in trans, bridging two kinases and "handcuffing" them together to reinforce the dimer, instead of keeping them away from each other.[42] Further modes of 14-3-3 interactions with c-Raf also exist, but their role is not well known.[43]

Dimerisation is another important mechanism for c-Raf activity regulation and required for Raf activation loop phosphorylation. Normally, only the "open" kinase domains participate in dimerisation. Unlike B-Raf, that readily forms homodimers with itself, c-Raf prefers heterodimerisation with either B-Raf or KSR1.[citation needed] Homodimers and heterodimers all behave similarly.[33] The B-Raf homodimer kinase domain structure clearly shows that the activation loops (that control the catalytic activity of all known protein kinases) are positioned in an active-like conformation in the dimer. This is due to an allosteric effect of the other molecule binding to the "back" side of the kinase; such dimers are symmetric and have two, partially active catalytic sites. At this stage, the activity of Raf kinases is low, and unstable.

The activation cycle of mammalian Raf proteins, exemplified by B-Raf (a greatly simplified overview, not showing all steps).[35][36]

To achieve full activity and stabilize the active state, the activation loop of c-Raf needs to be phosphorylated. The only kinases currently known to perform this act are the Raf family kinases themselves. But some other kinases, such as PAK1 can phosphorylate other residues near the kinase domain of c-Raf: the precise role of these auxiliary kinases is unknown. In the context of c-Raf, both c-Raf and KSR1 are needed for the "transphosphorylation" step. Due to the architecture of the dimers, this phosphorylation can only take place in trans (i.e. one dimer phosphorylates another, in a four-membered transitional complex).[44] By interacting with conserved Arg and Lys residues in the kinase domain, the phosphorylated activation loops shift conformation and become ordered, permanently locking the kinase domain into a fully active state until dephosphorylated. The phosphorylated activation loops also render the kinase insensitive to the presence of its autoinhibitory domain.[45] KSRs cannot undergo this last step as they miss any phosphorylatable residues in their activation loops. But once c-Raf is fully activated, there is no further need to do so: active Raf enzymes can now engage their substrates.[46] Like most protein kinases, c-Raf has multiple substrates. BAD (Bcl2-atagonist of cell death) is directly phosphorylated by c-Raf,[47] along with several types of adenylate cyclases,[48] myosin phosphatase (MYPT),[49] cardiac muscle troponin T (TnTc),[50] etc. The retinoblastoma protein (pRb) and Cdc25 phosphatase were also suggested as possible substrates.[51]

The most important targets of all Raf enzymes are MKK1(MEK1) and MKK2(MEK2). Although the structure of the enzyme-substrate complex c-Raf:MKK1 is unknown, it can be precisely modelled after the KSR2:MKK1 complex.[33] Here no actual catalysis takes place, but it is thought to be highly similar to the way Raf binds to its substrates. The main interaction interface is provided by the C-terminal lobes of both kinase domains; the large, disordered, proline-rich loop unique to MKK1 and MKK2 also plays an important role in its positioning to Raf (and KSR).[52] These MKKs become phosphorylated on at least two sites in their activation loops upon binding to Raf: this will activate them too. The targets of the kinase cascade are ERK1 and ERK2, that are selectively activated by MKK1 or MKK2. ERKs have numerous substrates in cells; they are also capable of translocating into the nucleus to activate nuclear transcription factors. Activated ERKs are pleiotropic effectors of cell physiology and play an important role in the control of gene expression involved in the cell division cycle, cell migration, inhibition of apoptosis, and cell differentiation.

Associated human diseases

[edit]

Hereditary gain-of-function mutations of c-Raf are implicated in some rare, but severe syndromes. Most of these mutations involve single amino acid changes at one of the two 14-3-3 binding motifs.[53][54] Mutation of c-Raf is one of the possible causes of Noonan syndrome: affected individuals have congenital heart defects, short and dysmorphic stature and several other deformities. Similar mutations in c-Raf can also cause a related condition, termed LEOPARD syndrome (Lentigo, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Retarded growth, Deafness), with a complex association of defects.

Role in cancer

[edit]

Although c-Raf is very clearly capable of mutating into an oncogene in experimental settings, and even in a few human tumors,[55][56] its sister kinase B-Raf is the true major player in carcinogenesis in humans.[57]

B-Raf mutations

[edit]

Approximately 20% of all examined human tumor samples display a mutated B-Raf gene.[58] The overwhelming majority of these mutations involve the exchange of a single amino acid: Val 600 into Glu, and this aberrant gene product (BRAF-V600E) can be visualized by immunohistochemistry for clinical molecular diagnostics[59][60] The aberration can mimic the activation loop phosphorylation and - by jumping all control steps at normal activation - immediately render the kinase domain fully active.[61] Since B-Raf can also activate itself by homodimerisation and c-Raf by heterodimerisation, this mutation has a catastrophic effect by turning the ERK1/2 pathway constitutively active, and driving an uncontrolled process of cell division.[62]

As a therapeutic target

[edit]

Due to the importance of both Ras and B-Raf mutations in tumorigenesis, several Raf inhibitors were developed to combat cancer, especially against B-Raf exhibiting the V600E mutation. Sorafenib was the first clinically useful agent, that provides a pharmacological alternative to treat previously largely untreatable malignancies, such as renal cell carcinoma and melanoma.[63] Several other molecules followed up, such as Vemurafenib, Regorafenib, Dabrafenib, etc.

Unfortunately, ATP-competitive B-Raf inhibitors may have an undesired effect in K-Ras-dependent cancers: They are simply too selective for B-Raf. While they perfectly well inhibit B-Raf activity in case a mutant B-Raf is the primary culprit, they also promote homo- and heterodimerisation of B-Raf, with itself and c-Raf. This will actually enhance c-Raf activation instead of inhibiting it in case there is no mutation in any Raf genes, but their common upstream activator K-Ras protein is the one mutated.[27] This "paradoxical" c-Raf activation necessitates the need to screen for B-Raf mutations in patients (by genetic diagnostics) before starting a B-Raf-inhibitor therapy.[64]

List of interacting proteins

[edit]

C-Raf has been shown to interact with:

See also

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References

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Further reading

[edit]
[edit]
Revisions and contributorsEdit on WikipediaRead on Wikipedia

C-Raf

View on Grokipedia
from Grokipedia
c-Raf, also known as RAF1 or Raf-1, is a encoded by the RAF1 proto- on human , serving as a critical mediator in the RAS-RAF-MEK-ERK (MAPK) signaling cascade that regulates cellular processes such as proliferation, differentiation, survival, and . Originally identified in the as the cellular homolog of the v-raf viral , c-Raf functions downstream of Ras , where binding to active Ras-GTP recruits it to the plasma membrane for via a series of events and dimerization with other RAF family members like B-Raf. Structurally, the 648-amino-acid protein (~73 kDa) comprises an N-terminal regulatory domain with Ras-binding and cysteine-rich regions, a central hinge, and a C-terminal domain, allowing tight control through inhibitory intramolecular interactions that are relieved upon stimulation; recent cryo-EM studies as of 2025 have provided deeper insights into these dynamics. In physiological contexts, c-Raf integrates signals from receptor tyrosine kinases and G-protein-coupled receptors to phosphorylate and activate MEK1/2, which in turn stimulate ERK1/2 to influence and cytoskeletal dynamics, with studies in mice revealing its essential role in embryonic development and vascular integrity, as embryos lacking c-Raf die around E10.5-E12.5 due to defective placental and hematopoietic functions. Beyond normal signaling, c-Raf is implicated in oncogenesis, where it is frequently overexpressed in various cancers including , liver, and malignancies, facilitating tumor progression by amplifying RAS-driven signals even in the absence of direct RAF1 mutations, which are rare compared to those in B-RAF; emerging evidence also highlights RAF1 fusions and amplifications in some tumors. Its dysregulation also contributes to therapeutic resistance, such as paradoxical activation of the MAPK pathway in response to B-RAF inhibitors in wild-type cells, highlighting c-Raf's role in adaptive signaling networks.

History and Discovery

Initial Identification of Raf Kinases

The Raf kinase family was first identified through the discovery of the viral oncogene v-raf in 1983, transduced by the acutely transforming murine sarcoma retrovirus 3611-MSV. This retrovirus, isolated from a tumor, efficiently transformed NIH 3T3 fibroblasts upon with its proviral DNA, producing foci at a rate of approximately 4 per nanogram, and the recovered virus induced fibrosarcomas in inoculated mice. The v-raf was characterized as a novel sequence without close homology to previously known viral oncogenes, though its transforming potential suggested an initial association with activity, akin to other retroviral oncoproteins like v-src. This finding, reported by U.R. Rapp and colleagues, marked the initial recognition of the Raf family as key players in cellular transformation. The cellular homolog of v-raf, termed c-Raf (also known as c-Raf-1 or Raf-1), was subsequently identified in mammalian cells in the mid-1980s. In 1984, chromosomal mapping studies located the human c-raf locus on , confirming its distinction from other families and its conservation across species. The full coding of human c-raf was cloned and reported in 1986, revealing a 648-amino-acid protein with conserved motifs indicative of a , thus establishing its biochemical classification within the superfamily. This cellular counterpart demonstrated structural similarity to v-raf, particularly in its kinase domain, underscoring the retroviral oncogene's origin as a captured and altered version of a normal cellular involved in . Initial functional studies of Raf kinases in the late 1980s began to elucidate their role in signaling pathways. Experiments demonstrated that c-Raf undergoes rapid in response to growth factors such as and , as well as upon expression of membrane-bound oncogenes like v-src, indicating activation within minutes of stimulation in transformed cells. Complementary work with temperature-sensitive mutants of avian retroviruses carrying related Raf-like oncogenes, such as v-mil in the MH2 virus, showed that shifting to permissive temperatures restored transforming activity and morphological changes in infected cells, linking Raf members to sustained proliferative signaling. These early investigations, building on the transforming properties observed with v-raf, positioned the Raf kinases as critical intermediaries in mitogenic signal relay from cell surface receptors to intracellular effectors.

Specific Characterization of c-Raf

The human c-Raf, also known as RAF1, was cloned as a cDNA from a fetal liver library, revealing an encoding a 648-amino acid protein containing a domain. The activity of c-Raf was demonstrated in 1991 through assays, in which immunoprecipitated c-Raf from stimulated cells phosphorylated exogenous substrates including basic protein, confirming its role as an active . Early subcellular localization studies utilizing in revealed that c-Raf is predominantly distributed in the under basal conditions but translocates to the plasma membrane upon stimulation with growth factors such as colony-stimulating factor 1, highlighting its dynamic localization in response to signaling cues. Mapping studies in the early , building on initial data, confirmed the chromosomal assignment of the RAF1 gene to the 3p25 region in humans, positioning it within a locus implicated in certain cancers and providing a genetic framework for further functional analyses.

Structure

Domain Organization

c-Raf, also known as RAF1, is a 648-amino-acid serine/threonine that consists of an N-terminal regulatory region spanning residues 1-257 and a C-terminal domain encompassing residues 306-648. The N-terminal region maintains the protein in an autoinhibited state through intramolecular interactions with the domain, while the C-terminal domain harbors the catalytic activity essential for in the MAPK/ERK pathway. The regulatory domain includes several key components that mediate interactions with upstream regulators. The conserved region 1 (CR1), corresponding to residues 51-131, contains a zinc finger-like structure that facilitates binding to GTP-bound Ras, thereby recruiting c-Raf to the plasma membrane for activation. Adjacent to CR1 is the conserved region 2 (CR2), a serine-rich segment from residues 256-269, which serves as a binding site for 14-3-3 proteins upon phosphorylation at serine 259, stabilizing the inactive conformation. The kinase domain adopts a conserved bilobal typical of eukaryotic protein kinases, with an N-lobe involved in ATP binding and a C-lobe responsible for substrate recognition. The ATP-binding site features a glycine-rich loop (G-loop) at residues 377-382, which positions the for phosphoryl transfer. The activation loop, spanning residues 491-502, includes critical sites serine 497 and serine 499, which are targeted by and modulate catalytic competence upon modification. Connecting the regulatory and kinase domains is a flexible inter-domain linker region (residues 258-305), which plays a role in autoinhibition by allowing the N-terminal region to occlude the kinase and can influence dimerization interfaces.

Structural Dynamics and Recent Insights

In the autoinhibited state of c-Raf, the N-lobe of the kinase domain engages with the regulatory domain, particularly through interactions involving the cysteine-rich domain (CRD), which nestles against the kinase domain and helps bury the activation segment in an autoinhibitory conformation. This configuration locks the C-helix in an outward, inactive position, as revealed by cryo-EM structures at 3.4 Å resolution. Earlier NMR studies from 2017 provided initial insights into the dynamic regulatory interactions contributing to this autoinhibition, while a 2022 further corroborated the burial of the activation segment in related Raf family members. Dimerization-induced of c-Raf involves the formation of a side-to-side interface between kinase domains, which relieves autoinhibition by rearranging the C-helix into an active inward position and exposing the dimerization surface. This process is particularly asymmetric in c-Raf/B-Raf heterodimers, where one protomer adopts a more active conformation to drive the partner's , as detailed in 2022 cryo-EM studies of the monomer-to-dimer transition. The side-to-side dimer interface, often mediated by 14-3-3 binding to phosphorylated serine residues, promotes back-to-back alignment in the active state, enabling subsequent MEK phosphorylation. Recent cryo-EM structures from 2025 of the c-Raf/MEK1/14-3-3 ternary complex, resolved at 3.4 Å for the autoinhibited state, illustrate how 14-3-3 stabilizes the inactive state by binding the CR2 motif, specifically the phosphorylated Ser259 site, which sequesters the kinase domain and prevents dimerization. In this complex, the 14-3-3 dimer engages both segments (pSer259 and pSer621) on opposite sides, maintaining the CRD in a cradle-like position that blocks regulatory domain release. These structures highlight two open-monomer intermediates where pSer259 is released, facilitating and progression toward activation. The Hsp90 chaperone complex, known as the RHC (Raf--Cdc37), plays a critical role in stabilizing the nascent kinase domain of c-Raf during folding, as shown in structural data. Cryo-EM analysis at 3.7 resolution reveals an asymmetric interaction where the unfolded N-lobe β5-strand of c-Raf threads through the luminal cavity, engaging via van der Waals contacts with 's src-loops and helical hairpins, while Cdc37 bridges the N- and C-lobes to the chaperone. This configuration ensures proper maturation of the kinase domain, with molecular simulations confirming strong electrostatic stabilization (e.g., -171.11 kcal/mol for the pS13-K405 interaction).

Evolutionary Relationships

Conservation Across Species

c-Raf, encoded by the RAF1 gene, displays high sequence conservation across vertebrate species, particularly in its kinase domain. The human Raf1 protein shares 98.9% amino acid sequence identity with its ortholog, reflecting strong evolutionary preservation of core catalytic functions. This high similarity extends to the kinase domain, where identity exceeds 90% among vertebrates, enabling conserved roles in . In contrast, the N-terminal regulatory domain shows lower conservation, approximately 70%, allowing for species-specific regulatory nuances while maintaining essential Ras-binding motifs. In , c-Raf orthologs exhibit moderate sequence similarity, underscoring functional homology in Ras-MAPK signaling. The Raf homolog, known as Polehole (Raf or D-Raf), shares about 40% overall identity with human c-Raf, with higher conservation in the kinase domain. This ortholog is essential for torso receptor tyrosine kinase (RTK) signaling during embryonic patterning. Similarly, the lin-45 gene encodes a Raf homolog required for vulval induction downstream of let-60 Ras, displaying significant sequence similarity in key domains despite overall lower identity compared to vertebrates. Functional conservation is evident from genetic studies across species. In mice, targeted disruption of the Raf1 gene results in embryonic lethality around mid-gestation, characterized by growth retardation and defects in placental and fetal liver development, as reported in late analyses. This phenotype mirrors the developmental defects observed in upon Raf loss, including disruptions in where the EGFR/Ras/Raf pathway specifies fates. Such parallels highlight the preserved necessity of c-Raf for RTK-mediated signaling in . The evolutionary roots of c-Raf trace to the tyrosine kinase-like (TKL) family, which emerged in the last eukaryotic common ancestor approximately 1.5 billion years ago, with partial homologs appearing in early eukaryotes but no direct Raf kinases in . This ancient origin supports the broad conservation of Raf-mediated pathways from unicellular organisms to complex metazoans.

Relations to Other Raf Family Members

The Raf family of serine/ in humans consists of three isoforms: c-Raf (encoded by RAF1), B-Raf (encoded by BRAF), and A-Raf (encoded by ARAF). These genes are located on different chromosomes: RAF1 on 3p25.2, BRAF on 7q34, and ARAF on Xp11.2. Despite these distinct genomic positions, the isoforms share approximately 75% identity in their kinase domains, while exhibiting notable differences in their N-terminal regulatory regions that influence activation and specificity. In terms of intrinsic activity, c-Raf displays the lowest basal activity among the isoforms and typically requires dimerization for full , whereas B-Raf possesses the highest basal activity and can function as a under certain conditions. A-Raf exhibits the weakest overall activity toward MEK substrates and shows tissue-restricted expression, predominantly in urogenital tissues. In contrast, c-Raf maintains the broadest expression pattern across tissues, enabling its involvement in diverse cellular contexts.48650-7/fulltext) Functional redundancy exists among the isoforms, particularly through the formation of c-Raf/B-Raf heterodimers, which are prevalent in Ras-mediated MAPK signaling and enhance pathway activation. studies in mice reveal compensatory mechanisms; for instance, c-Raf can partially compensate for B-Raf loss in cardiac development, mitigating some hypertrophic responses despite B-Raf's dominant role in certain contexts.01054-4.pdf) The evolutionary divergence of the Raf family stems from events associated with the two rounds of whole-genome duplication (2R hypothesis) early in evolution, approximately 500 million years ago, leading to the three paralogs observed in mammals; c-Raf has retained the most ubiquitous expression profile among them.

Regulation of Activity

Activation Pathways

The activation of c-Raf, a serine/ central to the MAPK/ERK pathway, is initiated primarily through Ras-dependent recruitment to the plasma membrane. GTP-bound Ras interacts with high affinity (approximately 10 nM) to the cysteine-rich domain (CR1) of c-Raf, specifically via the (RBD), which facilitates the translocation of cytosolic c-Raf to the membrane. This binding involves key residues R89 and Y91 within the CR1 region of c-Raf, enabling the disruption of autoinhibitory interactions that maintain c-Raf in an inactive state. Consequently, Ras recruits c-Raf to lipid rafts enriched in signaling molecules, positioning it for subsequent activating events. Upstream signals from receptor , such as EGFR, trigger the GDP-to-GTP exchange on Ras via guanine nucleotide exchange factors (GEFs), thereby activating the Ras-c-Raf interaction. This recruitment enables Src family to c-Raf at 341 (Y341) within its domain, a critical step that enhances activity and promotes downstream signaling. Src-mediated at Y341 occurs in a Ras-dependent manner, stabilizing an open conformation conducive to further activation. Scaffold proteins like kinase suppressor of Ras 1 and 2 (KSR1/2) further augment c-Raf activation by assembling a multiprotein complex at the plasma membrane, recruiting c-Raf alongside Ras, MEK, and other components to increase signaling efficiency. Discovered in mammalian systems in , KSR1 acts as a docking platform that coordinates these interactions without intrinsic kinase activity, thereby amplifying the localized activation of c-Raf. This scaffold-mediated recruitment ensures spatial organization, preventing cross-talk and optimizing the phosphorylation cascade. Dimerization represents another key mechanism, where c-Raf forms homodimers or heterodimers with B-Raf through a conserved interface in their domains. Studies using fluorescence resonance energy transfer () in the 2010s demonstrated that this dimerization, induced by Ras binding, enables allosteric of the receiver protomer independent of ATP binding, thereby boosting catalytic efficiency. The asymmetric nature of these dimers allows one subunit to allosterically relieve autoinhibition in the partner, a process essential for full competence.

Inhibitory and Feedback Mechanisms

One key inhibitory mechanism of c-Raf involves the binding of 14-3-3 proteins, which is facilitated by at serine 259 (S259) in the conserved region 2 (CR2) and serine 621 (S621) in the conserved region 3 (CR3). This binding stabilizes the autoinhibited monomeric conformation of c-Raf, preventing premature and ensuring signaling fidelity by antagonizing recruitment to the plasma membrane in response to weak stimuli. Recent cryo-electron microscopy (cryo-EM) structures of the CRAF₂/14-3-3₂ complex at 3.4 Å resolution reveal that the dimeric 14-3-3 proteins bridge the CR2 and CR3 regions, enforcing an autoinhibitory state that is disrupted upon RAS-mediated membrane recruitment. A 2025 study further provides cryo-EM structures of CRAF/MEK1/14-3-3 complexes at resolutions up to 2.3 Å, elucidating autoinhibited and open-monomer states and additional regulatory features. Negative regulation of c-Raf also occurs through inhibitory at specific sites, including S259 and threonine 401 (T401). of S259 by (PKA) in response to elevated cAMP levels directly suppresses c-Raf activity, rendering it resistant to and correlating with deactivation of downstream ERK signaling. Similarly, at T401 inhibits c-Raf function by disrupting key protein interactions within the MAPK cascade. For to proceed, of these sites, particularly S259, is essential and is mediated by 2A (PP2A), which associates with c-Raf to promote membrane localization and relieve inhibition. Feedback inhibition further fine-tunes c-Raf activity through downstream ERK-mediated . Activated ERK phosphorylates c-Raf at T401 (and homologous sites such as S289, S296, and S301), which dampens pathway signaling by promoting dissociation from upstream activators like RAS. This negative feedback loop also targets the kinase suppressor of RAS (KSR) scaffold, where ERK at sites like T260, T274, S320, and S443 disrupts the KSR-c-Raf complex, attenuating signal transmission to MEK. Studies from the in cellular models, including fibroblasts, demonstrated this ERK-dependent as a rapid mechanism to limit sustained MAPK activation following stimulation. To prevent prolonged signaling, c-Raf undergoes proteasomal degradation via ubiquitination, particularly after extended activation periods. The E3 ubiquitin ligase CHIP targets misfolded or kinase-inactive forms of c-Raf for polyubiquitination, leading to their clearance by the 26S ; this process is autophosphorylation-dependent at S621, as unphosphorylated c-Raf is rapidly degraded to maintain . inhibitors such as MG132 confirm this pathway by stabilizing c-Raf levels, highlighting its role in terminating signaling to avoid aberrant cellular responses.

Biological Functions

Role in MAPK/ERK Signaling

c-Raf, also known as Raf-1, serves as a key kinase (MAP3K) in the canonical Ras-Raf-MEK-ERK signaling cascade, where it directly phosphorylates and activates MEK1/2 by targeting serine residues at positions 218 and 222 in their activation loops. This phosphorylation event, characterized by a Michaelis constant (Km) of approximately 0.8-1 μM for MEK as substrate, enables MEK1/2 to subsequently phosphorylate and activate ERK1/2 at and residues in their TEY motif. Activated ERK1/2 then translocates to the nucleus to phosphorylate transcription factors such as Elk-1, thereby regulating programs involved in and differentiation. In response to extracellular stimuli like growth factors including (PDGF) and (EGF), c-Raf integrates signals primarily through upstream activation by GTP-bound Ras at the plasma membrane, leading to rapid amplification of mitogenic responses. This activation typically manifests as transient pulses of ERK , peaking within 5-10 minutes post-stimulation before returning to baseline due to feedback mechanisms, ensuring precise temporal control of downstream effects. Among Raf isoforms, c-Raf predominantly transduces stress-related signals, such as those induced by tumor necrosis factor-α (TNF-α), whereas B-Raf is the primary mediator of growth factor-driven ERK activation; notably, c-Raf/B-Raf heterodimers enhance signaling efficiency by promoting allosteric activation and increased output within the cascade. Systems-level modeling using flux control analysis has revealed that only about 10-20% activation of the total cellular c-Raf pool is sufficient to achieve maximal ERK signaling flux, underscoring the pathway's ultrasensitive response and the isoform's role as a tunable rather than a strict rate-limiter. This quantitative aspect highlights how events, such as those at c-Raf's serine 338 and 341 residues, fine-tune its contribution to overall pathway dynamics.

Involvement in Other Cellular Processes

Beyond its canonical role in the MAPK/ERK pathway, c-Raf contributes to the regulation of through post-translational modifications of pro-apoptotic proteins. Specifically, of c-Raf stimulates the of the BH3-only protein BAD at serine 112 (Ser112), which promotes its sequestration in the by 14-3-3 proteins, thereby preventing BAD from binding and inhibiting the anti-apoptotic protein at the mitochondria. This mechanism enhances cell survival in a kinase-dependent manner via downstream effectors like RSK. Studies in cultured cerebellar granule neurons have demonstrated that growth factor-induced c-Raf leads to BAD at Ser112 and Ser136, cooperatively inactivating BAD and protecting neurons from induced by trophic factor withdrawal. c-Raf also participates in cytoskeletal organization through direct protein-protein interactions independent of its activity toward MEK. In particular, c-Raf binds to Rho-associated α (Rok-α, also known as ROCK1), inhibiting its activity and thereby modulating RhoA signaling to facilitate proper dynamics. This interaction is essential for the formation of stress fibers in fibroblasts, as evidenced by studies showing that c-Raf-deficient embryonic fibroblasts exhibit hyperactivation of Rok-α, resulting in excessive cortical bundling and impaired stress fiber assembly; restoring c-Raf expression or inhibiting Rok-α reverses this defect and promotes stress fiber formation and cell spreading. These MEK-independent effects highlight c-Raf's function in maintaining cytoskeletal integrity during and . In the context of angiogenesis, c-Raf supports vascular development by influencing endothelial cell behavior, including the regulation of vascular endothelial growth factor (VEGF) expression through crosstalk with NF-κB signaling. In mouse models of growth plate maturation, conditional deletion of c-Raf in chondrocytes disrupts VEGF-A secretion, delaying endothelial invasion and vascularization, underscoring c-Raf's role in coordinating angiogenic cues. Although primarily studied in non-endothelial compartments, emerging evidence from endothelial cell lines indicates that c-Raf activation enhances NF-κB-mediated transcription of VEGF in response to inflammatory stimuli, fostering autocrine loops that promote endothelial proliferation and tube formation in vitro. c-Raf links to metabolic regulation in hepatic cells by integrating with insulin signaling pathways that control homeostasis. Through the Raf-1/MEK/ERK/p90RSK cascade, c-Raf indirectly modulates activity by promoting the and inactivation of glycogen synthase kinase-3 (GSK-3), which otherwise inhibits via phosphorylations at multiple sites. This effect is particularly relevant in liver hepatocytes, where stimuli like AICAR activate c-Raf to enhance GSK-3 inhibition, thereby increasing synthesis in coordination with insulin-induced Akt signaling, though the pathway's contribution is context-dependent and secondary to PI3K/Akt.

Disease Associations

Non-Cancerous Disorders

c-Raf, encoded by the RAF1 gene, has been implicated in several non-cancerous disorders through its dysregulation in the MAPK/ERK signaling pathway, particularly in genetic developmental conditions known as and in inflammatory and neurological pathologies. Gain-of-function mutations in RAF1 lead to hyperactivation of downstream effectors, disrupting normal cellular processes during development and immune responses. In , a characterized by facial dysmorphology, , and congenital heart defects, rare RAF1 mutations cause gain-of-function effects that enhance ERK signaling, resulting in and other cardiac abnormalities. For instance, the S257L mutation in the CR2 domain of c-Raf impairs inhibitory at serine 259, promoting constitutive activity and leading to severe cardiac defects in affected individuals. These mutations were first identified in 2007, and RAF1 variants account for approximately 3-17% of cases, with a notable ~5% prevalence in cohorts lacking PTPN11 mutations. Patients with RAF1-associated exhibit a higher incidence of (up to 95%) compared to other genetic subtypes. Similarly, activating variants in RAF1 contribute to cardio-facio-cutaneous (CFC) syndrome, another involving ectodermal, cardiac, and craniofacial anomalies that impair postnatal development. The P261Q variant, located in the CR2 domain, exemplifies these changes by reducing autoinhibitory interactions and elevating MAPK pathway activity, which disrupts cellular proliferation and differentiation during embryogenesis. RAF1 mutations represent 10-15% of cases across , including CFC, where they are less common than BRAF variants but still drive overlapping phenotypes like and . These genetic alterations highlight c-Raf's conserved role in developmental signaling, as seen in evolutionary studies of MAPK conservation. Beyond genetic disorders, c-Raf participates in inflammatory responses, particularly in TLR4-mediated signaling that exacerbates sepsis. In mouse models of lipopolysaccharide (LPS)-induced sepsis, c-Raf activation downstream of TLR4 promotes phosphorylation of MEK and ERK, culminating in excessive cytokine release such as TNF-α and IL-6, which drives systemic inflammation and organ failure. Studies from the 2010s using Raf inhibitors like sorafenib demonstrated reduced cytokine production and improved survival in LPS-challenged mice, underscoring c-Raf's role in amplifying the innate immune response to bacterial endotoxins. This pathway's overactivation contributes to the cytokine storm observed in human sepsis, linking c-Raf to acute inflammatory disorders. Neurological associations involve c-Raf overactivation in models, where amyloid-β (Aβ) peptides induce Ras activation, leading to c-Raf-mediated ERK signaling and subsequent hyper. In Aβ-exposed neuronal cultures and transgenic mouse models, this cascade activates GSK-3β, a key , promoting formation and synaptic dysfunction. Inhibition of the Ras-Raf-ERK pathway reduces at disease-relevant sites like Ser396/404, suggesting c-Raf as a contributor to Aβ-driven neurodegeneration without directly causing oncogenesis.

Oncogenic Roles and Alterations

c-Raf, encoded by the RAF1 gene, contributes to oncogenesis primarily through hyperactivation rather than frequent direct mutations, often amplifying MAPK/ERK signaling in various cancers. Amplification and copy number gains of RAF1 occur in approximately 16% of cutaneous melanomas according to (TCGA) data, with higher rates up to 29% observed in some cohorts of metastatic samples, and these alterations are associated with worse . In lung cancers, RAF1 alterations including amplifications are less common, detected in about 2-3% of non-small cell lung carcinoma cases in pan-cancer analyses, yet overexpression of c-Raf correlates with disease progression across multiple tumor types. Such genomic gains lead to elevated c-Raf protein levels, enhancing tumor and survival independent of its kinase activity in certain contexts. Activating in c-Raf are rare in cancers, occurring in less than 1% of cases overall due to its intrinsically low basal activity compared to B-Raf. In , point in the c-Raf domain, such as those mimicking sites like S338, are infrequent, comprising around 2% of alterations in some subtypes, and are more prevalent in tumors with concurrent RAS where they potentiate downstream signaling. These typically enhance c-Raf's responsiveness to upstream RAS activation, promoting oncogenic transformation in RAS-mutant backgrounds rather than acting as primary drivers. c-Raf engages in significant with other oncogenes, amplifying malignant phenotypes through integrated pathway activation. In NRAS-driven melanomas, c-Raf serves as the primary effector of NRAS, enhancing PI3K/AKT signaling to support tumor growth and resistance to targeted therapies. Additionally, c-Raf compensates for BRAF inhibition by forming heterodimers with wild-type or B-Raf, leading to paradoxical ERK activation that drives resistance in BRAF- cancers. Recent studies highlight c-Raf's evolving role in KRAS- pancreatic cancer, where it promotes tumor invasion through both ERK-dependent and independent mechanisms, such as kinase-independent scaffolding functions that sustain pro-survival signaling. A 2023 review emphasizes c-Raf's context-specific contributions in KRAS-driven malignancies, underscoring its potential as a therapeutic vulnerability in these aggressive tumors.

Therapeutic Targeting

Development of Inhibitors

The development of c-Raf inhibitors includes early multikinase agents like , a type II ATP-competitive inhibitor approved by the FDA in 2005 for advanced , which binds the kinase's conserved ATP-binding pocket and extends into an allosteric pocket to stabilize the inactive DFG-out conformation, with an of approximately 6 nM for c-Raf while also inhibiting B-Raf (IC50 ~22 nM) and other kinases such as VEGFR2, resulting in a pan-Raf profile with notable off-target effects that limit selectivity. Subsequent advancements in type II inhibitors include compounds that further stabilize the inactive DFG-out conformation of the domain. PLX-4032 (), clinically approved for BRAF-mutant , represents this approach but paradoxically induces c-Raf in wild-type cells through enhanced Raf dimerization and of the MAPK pathway. To circumvent issues with orthosteric binding and paradoxical , researchers have explored allosteric modulators that target the Ras-binding site within the CR1 regulatory domain of c-Raf, disrupting upstream recruitment by active Ras-GTP. Preclinical compounds developed in the exemplify this strategy by binding to interfaces that prevent Ras-c-Raf interaction, thereby inhibiting without directly engaging the domain. More recently, proteolysis-targeting chimeras (PROTACs) have emerged as a degradation-based modality for c-Raf, recruiting ubiquitin ligases to induce -mediated proteasomal breakdown. For example, MS934, a VHL-recruiting MEK1/2 PROTAC, collaterally degrades CRAF in KRAS-mutant cells, offering a complementary mechanism to traditional inhibition by eliminating the protein entirely rather than merely blocking its activity.

Clinical Applications and Challenges

Sorafenib, a multikinase inhibitor targeting c-Raf among other kinases, received FDA approval in 2007 for the treatment of unresectable hepatocellular carcinoma (HCC), based on the SHARP trial demonstrating improved overall survival from 7.9 to 10.7 months compared to placebo. The objective response rate in this trial was approximately 2%, primarily attributed to Raf pathway inhibition, though the drug's broader effects on angiogenesis also contribute. Similarly, sorafenib was approved by the FDA in 2005 for advanced renal cell carcinoma (RCC), where it extended progression-free survival to 5.5 months versus 2.8 months with placebo in the TARGET trial, with c-Raf inhibition playing a key role in suppressing tumor proliferation. In BRAF-mutant , combination therapies incorporating c-Raf modulation have shown enhanced efficacy; for instance, the BRAF inhibitor combined with the improved median to 14.9 months in the phase III COLUMBUS trial, compared to 7.3 months with monotherapy, by mitigating paradoxical c-Raf activation. These 2018 results, with updates through the 2020s confirming durable benefits, highlight how dual inhibition addresses c-Raf-dependent resistance in BRAF V600-mutant settings. A major clinical challenge is resistance to Raf-targeted therapies, often arising from feedback reactivation of c-Raf dimers following B-Raf inhibition, a mechanism identified in the 2010s that sustains MAPK signaling in and other cancers. RAS mutations further predict poor response to c-Raf inhibitors by enhancing wild-type Raf dimerization and pathway reactivation, limiting efficacy in RAS-mutant tumors. Emerging strategies as of 2025 target complexes that regulate c-Raf activation, with structural studies revealing potential disruption sites to overcome dimer-mediated resistance. Ongoing preclinical efforts include development of PET imaging probes for pan-Raf kinases to better select patients and monitor therapeutic engagement, as well as type I pan-RAF inhibitors like ELV-3111 that combine safely with MEK inhibitors for enhanced activity in NRAS and BRAF mutant cancers.

Protein Interactions

Core Binding Partners

c-Raf, also known as Raf-1, interacts directly with members of the Ras family of small , including H-Ras, K-Ras, and N-Ras, primarily through its Ras-binding domain (RBD) within the conserved region 1 (CR1). These GTP-bound Ras proteins bind to the RBD with high affinity, characterized by a (Kd) of approximately 20 nM for H-Ras, facilitating the recruitment and translocation of cytosolic c-Raf to the plasma membrane where activation occurs. This interaction is essential for c-Raf's membrane localization and subsequent signaling, and the Ras-c-Raf interface has been extensively studied in over 100 publications, highlighting key residues such as Ras Q61 and Y32 that contribute to specificity and binding stability. 14-3-3 proteins, particularly isoforms σ and ζ, bind to phosphorylated residues on c-Raf, including serine 259 (S259) and serine 621 (S621), thereby inhibiting its kinase activity by maintaining an autoinhibited conformation. This binding was first identified in the mid-1990s through two-hybrid screens and co-immunoprecipitation assays, revealing that 14-3-3 association with the N-terminal regulatory domain of c-Raf prevents premature activation and promotes cytoplasmic retention. Pulldown experiments from that era confirmed the phosphorylation-dependent nature of these interactions, with S259 phosphorylation by kinases like AKT enhancing 14-3-3 binding to block Ras recruitment. As the primary substrate of c-Raf, MEK1 and MEK2 interact via a docking motif known as the D-domain on MEK, which engages the domain of c-Raf to position it for efficient at activation sites S217 and S221. The Michaelis constant (Km) for this reaction is approximately 0.5-1 μM, reflecting moderate substrate affinity that supports rapid signal propagation in the MAPK pathway. Structural studies have delineated the D-domain as a basic stretch of residues on MEK that binds an acidic patch on c-Raf, ensuring specificity in the kinase-substrate complex. c-Raf stability is maintained through interaction with the chaperone , mediated by the co-chaperone Cdc37, which binds the kinase domain of immature c-Raf to prevent degradation and facilitate maturation. Co-immunoprecipitation studies have demonstrated that disruption of the -Cdc37-c-Raf complex leads to ubiquitination and proteasomal degradation of c-Raf, underscoring 's role in stabilizing the protein under physiological conditions. This binary interaction, often observed in early biosynthetic stages, ensures proper folding without directly impacting catalytic activity once c-Raf is fully assembled.

Regulatory Complexes

c-Raf, also known as RAF1, is regulated through dynamic protein complexes that control its activation, localization, and signaling output in the MAPK/ERK pathway. These complexes primarily involve upstream activators like RAS, scaffolding proteins such as KSR, regulatory adapters like 14-3-3, and inhibitory factors including RKIP, which collectively modulate c-Raf's autoinhibited state, membrane recruitment, and dimerization. Structural studies have revealed that c-Raf's N-terminal regulatory region, comprising the and cysteine-rich domain (CRD), interacts with these partners to relieve intramolecular inhibition and facilitate domain activation. A central regulatory complex forms upon RAS-GTP binding to c-Raf's , which recruits c-Raf to the plasma membrane and disrupts autoinhibitory interactions between the N-terminal region and the domain. This RAS-c-Raf engagement is stabilized by the CRD's membrane interactions and is essential for subsequent dimerization with other RAF isoforms or KSR. Cryo-EM structures show that in the autoinhibited state, RAS binding initiates a conformational shift, exposing sites for and enabling c-Raf's transition to an open ready for . Additionally, the scaffold protein KSR1 assembles a multiprotein complex including c-Raf, MEK, and ERK, promoting efficient ; KSR1's pseudokinase domain heterodimerizes with c-Raf in a side-to-side manner, allosterically positioning c-Raf's catalytic αC for MEK . The 14-3-3 proteins form a critical regulatory complex with c-Raf by binding phosphorylated serine residues (pSer259 in the N-region and pSer621 in the kinase domain), stabilizing the autoinhibited monomer and preventing premature dimerization. Upon signaling, dephosphorylation of pSer259 by the SHOC2-MRAS-PP1C phosphatase complex releases this inhibition, allowing 14-3-3 to reorient and bridge two c-Raf protomers via their pSer621 sites, thereby promoting active back-to-back dimers. This 14-3-3-mediated dimerization is further supported by N-terminal phosphorylation (e.g., at Ser338 and Tyr341 by PAK or Src kinases), which enhances complex stability and c-Raf activity. In parallel, the chaperone complex HSP90-CDC37 binds c-Raf to maintain its folding and stability, while associated PP5 dephosphorylates regulatory sites post-activation to facilitate signal termination. Inhibitory regulatory complexes also play key roles; for instance, Inhibitory Protein (RKIP) binds the N-terminal region of c-Raf (residues ~Tyr340-Lys615), forming a stable complex via hydrogen bonds and hydrophobic interactions at hotspots like Tyr340, Arg398, and RKIP's Lys80 and Trp84. This binding prevents c-Raf at activating sites (Ser338, Tyr340/341) and disrupts c-Raf-MEK association, thereby suppressing MAPK signaling. simulations indicate a binding free energy of approximately -174 kJ/mol for the RKIP-c-Raf complex, underscoring its potency. Scaffolds like CNK1 can either enhance or inhibit c-Raf activation depending on context, often by modulating Src-mediated within these assemblies. Overall, these complexes ensure precise spatiotemporal control of c-Raf, integrating positive and negative inputs to fine-tune cellular responses.

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