BRAF (gene)

Source: Wikipedia, the free encyclopedia.
BRAF
Gene ontology
Molecular function
Cellular component
Biological process
Sources:Amigo / QuickGO
Ensembl

ENSG00000157764

ENSMUSG00000002413

UniProt

P15056

P28028

RefSeq (mRNA)

NM_004333
NM_001354609
NM_001374244
NM_001374258

NM_139294

RefSeq (protein)

NP_647455

Location (UCSC)Chr 7: 140.72 – 140.92 MbChr 6: 39.58 – 39.7 Mb
PubMed search[3][4]
Wikidata
View/Edit HumanView/Edit Mouse

BRAF is a human

proto-oncogene B-Raf and v-Raf murine sarcoma viral oncogene homolog B, while the protein is more formally known as serine/threonine-protein kinase B-Raf.[5][6]

The B-Raf protein is involved in sending

cancers.[7]

Certain other inherited BRAF mutations cause birth defects.

Drugs that treat cancers driven by BRAF mutations have been developed. Two of these drugs,

fragment-based drug discovery.[9]

Function

Raf kinase family of growth signal transduction protein kinases. This protein plays a role in regulating the MAP kinase/ERKs signaling pathway, which affects cell division, differentiation, and secretion.[10]

Structure

B-Raf is a 766-

Ras-GTP-binding[11] self-regulatory domain, conserved region 2 (CR2), a serine-rich hinge region, and conserved region 3 (CR3), a catalytic protein kinase domain that phosphorylates a consensus sequence on protein substrates.[12] In its active conformation, B-Raf forms dimers via hydrogen-bonding and electrostatic interactions of its kinase domains.[13]

CR1

Conserved region 1 (CR1)

DAG-binding zinc finger motif that participates in B-Raf membrane docking after Ras-binding.[14][15]

CR2

Conserved region 2 (CR2) provides a flexible linker that connects CR1 and CR3 and acts as a hinge.[citation needed]

CR3

Figure 1: Inactive conformation of B-Raf kinase (CR3) domain. P-Loop (orange) hydrophobic interactions with activation loop (gray) residues that stabilize the inactive kinase conformation are shown with sticks. F595 (red) blocks the hydrophobic pocket where the ATP adenine binds (yellow). D576 (orange) is shown as part of the catalytic loop (magenta). Figure modified from PDB id 1UWH.

Conserved region 3 (CR3), residues 457–717,

substrate proteins.[16] The active site is the cleft between the two lobes, and the catalytic Asp576 residue is located on the C-lobe, facing the inside of this cleft.[14][16]

Subregions

P-Loop

The P-loop of B-Raf (residues 464–471) stabilizes the non-transferable phosphate groups of ATP during enzyme ATP-binding. Specifically, S467, F468, and G469 backbone amides hydrogen-bond to the β-phosphate of ATP to anchor the molecule. B-Raf functional motifs have been determined by analyzing the homology of PKA analyzed by Hanks and Hunter to the B-Raf kinase domain.[16]

Nucleotide-binding pocket

V471, C532, W531, T529, L514, and A481 form a hydrophobic pocket within which the adenine of ATP is anchored through Van der Waals attractions upon ATP binding.[16][18]

Catalytic loop

Residues 574–581 compose a section of the kinase domain responsible for supporting the transfer of the γ-phosphate of ATP to B-Raf's protein substrate. In particular, D576 acts as a proton acceptor to activate the nucleophilic hydroxyl oxygen on substrate serine or threonine residues, allowing the phosphate transfer reaction to occur mediated by base-catalysis.[16]

DFG motif

cation that stabilizes the β- and γ-phosphate groups of ATP, orienting the γ-phosphate for transfer.[16]

Activation loop

Residues 596–600 form strong hydrophobic interactions with the P-loop in the inactive conformation of the kinase, locking the kinase in its inactive state until the

activation loop is phosphorylated, destabilizing these interactions with the presence of negative charge. This triggers the shift to the active state of the kinase. Specifically, L597 and V600 of the activation loop interact with G466, F468, and V471 of the P-loop to keep the kinase domain inactive until it is phosphorylated.[17]

Enzymology

B-Raf is a

bimolecular nucleophilic substitution.[16][20][21][22]

Activation

Relieving CR1 autoinhibition

The kinase (CR3) domain of human

Raf kinase family. The CR1-Ras interaction is later strengthened through the binding of the cysteine-rich subdomain (CRD) of CR1 to Ras and membrane phospholipids.[12] Unlike A-Raf and C-Raf, which must be phosphorylated on hydroxyl-containing CR2 residues before fully releasing CR1 to become active, B-Raf is constituitively phosphorylated on CR2 S445.[23]
This allows the negatively charged phosphoserine to immediately repel CR1 through steric and electrostatic interactions once the regulatory domain is unbound, freeing the CR3 kinase domain to interact with substrate proteins.

CR3 domain activation

After the autoinhibitory CR1 regulatory domain is released, B-Raf's CR3

activation loop residues form hydrophobic interactions with the P-loop, stopping ATP from accessing its binding site. When the activation loop is phosphorylated, the negative charge of the phosphate is unstable in the hydrophobic environment of the P-loop. As a result, the activation loop changes conformation, stretching out across the C-lobe of the kinase domain. In this process, it forms stabilizing β-sheet interactions with the β6 strand. Meanwhile, the phosphorylated residue approaches K507, forming a stabilizing salt bridge to lock the activation loop into place. The DFG motif changes conformation with the activation loop, causing F595 to move out of the adenine nucleotide binding site and into a hydrophobic pocket bordered by the αC and αE helices. Together, DFG and activation loop movement upon phosphorylation open the ATP binding site. Since all other substrate-binding and catalytic domains are already in place, phosphorylation of the activation loop alone activates B-Raf's kinase domain through a chain reaction that essentially removes a lid from an otherwise-prepared active site.[17]

Mechanism of catalysis

Figure 2: Base-catalyzed nucleophilic attack of a serine/threonine substrate residue on the γ-phosphate group of ATP. Step 1: chelation of secondary magnesium ion by N581 and deprotonation of substrate Ser/Thr by D576. Step 2: nucleophilic attack of activated substrate hydroxyl on ATP γ-phosphate. Step 3: magnesium complex breaks down and D576 deprotonates. Step 4: release of products.

To effectively catalyze protein phosphorylation via the bimolecular substitution of serine and threonine residues with ADP as a leaving group, B-Raf must first bind ATP and then stabilize the transition state as the γ-phosphate of ATP is transferred.[16]

ATP binding

B-Raf binds ATP by anchoring the adenine nucleotide in a

carboxyl group balances this charge.[16][17]

Phosphorylation

Once ATP is bound to the B-Raf kinase domain, D576 of the catalytic loop activates a substrate hydroxyl group, increasing its nucleophilicity to kinetically drive the phosphorylation reaction while other catalytic loop residues stabilize the transition state (Figure 2). N581 chelates the divalent magnesium cation associated with ATP to help orient the molecule for optimal substitution. K578 neutralizes the negative charge on the γ-phosphate group of ATP so that the activated ser/thr substrate residue will not experience as much electron-electron repulsion when attacking the phosphate. After the phosphate group is transferred, ADP and the new phosphoprotein are released.[16]

Inhibitors

Since constitutively active B-Raf mutants commonly cause cancer (see Clinical Significance) by excessively signaling cells to grow, inhibitors of B-Raf have been developed for both the inactive and active conformations of the kinase domain as cancer therapeutic candidates.[17][18][19]

Sorafenib

phenyl
ring further prohibits DFG motif and activation loop movement to the active confermer via steric blockage.

BAY43-9006 (

affinity for the kinase domain. It then binds key activation loop and DFG motif residues to stop the movement of the activation loop and DFG motif to the active conformation. Finally, a trifluoromethyl phenyl moiety sterically blocks the DFG motif and activation loop active conformation site, making it impossible for the kinase domain to shift conformation to become active.[17]

The distal

carbonyl accepts a hydrogen bond from D594's backbone amide nitrogen to lock the DFG motif in place.[17]

The trifluoromethyl phenyl moiety cements the thermodynamic favorability of the inactive conformation when the kinase domain is bound to BAY43-9006 by sterically blocking the hydrophobic pocket between the αC and αE helices that the DFG motif and activation loop would inhabit upon shifting to their locations in the active conformation of the protein.[17]

Vemurafenib

Figure 4: Structures of Vemurafenib (right) and its precursor, PLX 4720 (left), two inhibitors of the active conformation of the B-Raf kinase domain

PLX4032 (Vemurafenib) is a V600 mutant B-Raf inhibitor approved by the FDA for the treatment of late-stage melanoma.[13] Unlike BAY43-9006, which inhibits the inactive form of the kinase domain, Vemurafenib inhibits the active "DFG-in" form of the kinase,[18][19] firmly anchoring itself in the ATP-binding site. By inhibiting only the active form of the kinase, Vemurafenib selectively inhibits the proliferation of cells with unregulated B-Raf, normally those that cause cancer.

Since Vemurafenib only differs from its precursor, PLX4720, in a

carbonyl of that residue instead, creating repulsion. Thus, Vemurafenib binds preferentially to the active state of B-Raf's kinase domain.[18][19]

Clinical significance

Mutations in the BRAF gene can cause disease in two ways. First, mutations can be inherited and cause birth defects. Second, mutations can appear later in life and cause cancer, as an oncogene.

Inherited mutations in this gene cause cardiofaciocutaneous syndrome, a disease characterized by heart defects, mental retardation and a distinctive facial appearance.[24]

Mutations in this gene have been found in cancers, including

brain tumors including glioblastoma and pleomorphic xanthoastrocytoma as well as inflammatory diseases like Erdheim–Chester disease.[10]

The V600E mutation of the BRAF gene has been associated with

Lynch syndrome to reduce the number of patients undergoing unnecessary MLH1 sequencing.[25][26]

Mutants

More than 30 mutations of the BRAF gene associated with human cancers have been identified. The frequency of BRAF mutations varies widely in human cancers, from more than 80% in

hairy cell leukaemia.[37] High frequency of BRAF V600E mutations have been detected in ameloblastoma, a benign but locally infiltrative odontogenic neoplasm.[38] The V600E mutation may also be linked, as a single-driver mutation (a genetic 'smoking gun') to certain cases of papillary craniopharyngioma development.[39]

Other mutations which have been found are R461I, I462S, G463E, G463V, G465A, G465E, G465V, G468A, G468E, G469R, N580S, E585K, D593V, F594L, G595R, L596V, T598I, V599D, V599E, V599K, V599R, V600K, A727V, etc. and most of these mutations are clustered to two regions: the glycine-rich P loop of the N lobe and the activation segment and flanking regions.[17] These mutations change the activation segment from inactive state to active state, for example in the previous cited paper it has been reported that the aliphatic side chain of Val599 interacts with the phenyl ring of Phe467 in the P loop. Replacing the medium-sized hydrophobic Val side chain with a larger and charged residue as found in human cancer(Glu, Asp, Lys, or Arg) would be expected to destabilize the interactions that maintain the DFG motif in an inactive conformation, so flipping the activation segment into the active position. Depending on the type of mutation the kinase activity towards MEK may also vary. Most of the mutants stimulate enhanced B-Raf kinase activity toward MEK. However, a few mutants act through a different mechanism because although their activity toward MEK is reduced, they adopt a conformation that activates wild-type C-RAF, which then signals to ERK.

BRAF-V600E

Main article: V600E
  • BRAFV600E mutation confers a poor prognosis in metastatic colorectal cancer. Upon targeted inhibition of BRAF and/or EGFR in BRAF V600E colorectal cancer, SRC kinases become activated in a systematic manner. Remarkably, concurrent targeting of SRC alongside BRAF and EGFR has demonstrated increased treatment effectiveness. The compensatory activation of SRC kinases is mediated by an autocrine prostaglandin E2 loop, which can be blocked using cyclooxygenase-2 (COX2) inhibitors. Notably, the simultaneous targeting of COX2 with BRAF and EGFR has shown significant potential in preclinical models, leading to a sustained suppression of tumor growth.[40]
  • BRAF V600E is a determinant of sensitivity to proteasome inhibitors. Vulnerability to proteasome inhibitors is dependent on persistent BRAF signaling, because BRAF-V600E blockade by PLX4720 reversed sensitivity to carfilzomib in BRAF-mutant colorectal cancer cells. Proteasome inhibition might represent a valuable targeting strategy in BRAF V600E-mutant colorectal tumors.[41]

BRAF inhibitors

As mentioned above, some pharmaceutical firms are developing specific inhibitors of mutated B-raf protein for anticancer use because BRAF is a well-understood, high yield target.[18][42] Vemurafenib (RG7204 or PLX4032) was licensed by the US Food and Drug Administration as Zelboraf for the treatment of metastatic melanoma in August 2011 based on Phase III clinical data. Improved survival was seen, as well as a response rate to treatment of 53%, compared to 7–12% with the former best chemotherapeutic treatment, dacarbazine.[43] In clinical trials, B-Raf increased metastatic melanoma patient chance of survival. In spite of the drug's high efficacy, 20% of tumors still develop resistance to the treatment. In mice, 20% of tumors become resistant after 56 days.[44] While the mechanisms of this resistance are still disputed, some hypotheses include the overexpression of B-Raf to compensate for high concentrations of Vemurafenib[44] and upstream upregulation of growth signaling.[45]

More general

B-Raf inhibitors include GDC-0879, PLX-4720, Sorafenib, dabrafenib and encorafenib
.

panRAF inhibitors

Belvarafenib is classified as a panRAF inhibitor. A panRAF inhibitor blocks the catalytic function of both proteins in the dimer.[46]

Interactions

BRAF (gene) has been shown to

interact
with:

References

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  4. ^ "Mouse PubMed Reference:". National Center for Biotechnology Information, U.S. National Library of Medicine.
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Further reading

External links

Public Domain This article incorporates

U.S. National Cancer Institute
. This article incorporates text from the United States National Library of Medicine, which is in the public domain.


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