View source for BRAF

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==Overview==
''BRAF'' is a human [[gene]] that makes a [[protein]] called B-Raf. The gene is also referred to as '''proto-oncogene B-Raf''' and '''v-Raf murine sarcoma viral oncogene homolog B1''', while the protein is more formally known as '''serine/threonine-protein kinase B-Raf'''.<ref name="pmid2284096">{{cite journal | author = Sithanandam G, Kolch W, Duh FM, Rapp UR | title = Complete coding sequence of a human B-raf cDNA and detection of B-raf protein kinase with isozyme specific antibodies | journal = Oncogene | volume = 5 | issue = 12 | pages = 1775–80 | year = 1990 | month = December | pmid = 2284096 | doi = | url = }}</ref><ref name="pmid1565476">{{cite journal | author = Sithanandam G, Druck T, Cannizzaro LA, Leuzzi G, Huebner K, Rapp UR | title = B-raf and a B-raf pseudogene are located on 7q in man | journal = Oncogene | volume = 7 | issue = 4 | pages = 795–9 | year = 1992 | month = April | pmid = 1565476 | doi = | url = }}</ref> 

==B-Raf Protein==
The B-Raf protein is involved in sending [[signal transduction|signals]] inside cells, which are involved in directing [[cell growth]]. In 2002, it was shown to be faulty ([[Mutation|mutated]]) in human [[cancers]].<ref name="pmid12068308">{{cite journal | author = Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A, Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C, Shipley J, Hargrave D, Pritchard-Jones K, Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL, Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ, Wooster R, Stratton MR, Futreal PA | title = Mutations of the BRAF gene in human cancer | journal = Nature | volume = 417 | issue = 6892 | pages = 949–54 | year = 2002 | month = June | pmid = 12068308 | doi = 10.1038/nature00766 | url = | issn = }}</ref>

Certain other inherited ''BRAF'' mutations cause birth defects. 

Drugs that treat cancers driven by ''BRAF'' have been developed. Two of these drugs, [[vemurafenib]]<ref>{{cite press release|url=http://www.gene.com/gene/news/press-releases/display.do?method=detail&id=13567 |title=FDA Approves Zelboraf (Vemurafenib) and Companion Diagnostic for BRAF Mutation-Positive Metastatic Melanoma, a Deadly Form of Skin Cancer |publisher=Genentech |accessdate=2011-08-17}}</ref> and [[dabrafenib]] are approved by FDA for treatment of late-stage melanoma. Vemurafenib was the first drug to come out of [[fragment-based drug discovery]].

==Biological Function==
The role of Raf proteins, like B-Raf is indicated in the center.]]B-Raf is a member of the [[Raf kinase]] family of growth [[signal transduction]] [[protein kinase]]s. This protein plays a role in regulating the [[Mitogen-activated protein kinase|MAP kinase]]/[[extracellular signal-regulated kinases|ERK]]s [[MAPK/ERK pathway|signaling pathway]], which affects [[cell division]], [[cellular differentiation|differentiation]], and secretion.<ref name="entrez">{{cite web | title = Entrez Gene: BRAF | url = http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=673| accessdate = }}</ref>

== Protein Structure and Function ==
B-Raf is a 766-[[amino acid]], regulated [[signal transduction]] [[serine/threonine-specific protein kinase]].  Broadly speaking, it is composed of three conserved [[Protein domain|domains]] characteristic of the [[Raf kinase| Raf kinase family]]: conserved region 1 (CR1), a [[Ras subfamily|Ras]]-[[Guanosine triphosphate|GTP]]-binding<ref name="pmid7855890">{{cite journal | author = Daum, G et al. | title = The ins and outs of Raf kinases | journal = Trends in Biochem. Sci. | volume = 19 | issue = 11 | pages = 474–80 | year = 1994 | month = November | pmid = 7855890 | doi = | url = | issn = }}</ref> self-regulatory domain, conserved region 2 (CR2), a [[serine]]-rich hinge region, and conserved region 3 (CR3), a catalytic [[protein kinase]] domain that [[Phosphorylation|phosphorylates]] a [[consensus sequence]] on protein substrates.<ref name="Cutler Autoinhibition">{{cite journal | author = Cutler, RE et al. | title = Autoregulation of the Raf-1 serine/threonine kinase | journal = PNAS | volume = 95 | issue = 16 | pages = 9214–9219 | year = 1998 | month = August | pmid = 9689060 | doi = | url = http://www.pnas.org/content/95/16/9214 | issn = | pmc=21318}}</ref>  In its active conformation, B-Raf forms dimers via [[Hydrogen bond|hydrogen-bonding]] and electrostatic interactions of its kinase domains.<ref name ="pmid23060265">{{cite journal | author = Bollag, G et al. | title = Vemurafenib: the first drug approved for BRAF-mutant cancer | journal = Nature Rev. Drug Discov. | volume = 11 | issue = 11 | pages = 873–886 | year = 2012 | month = November | pmid = 23060265 | doi = 10.1038/nrd3847 | url = http://www.ncbi.nlm.nih.gov/pubmed/?term=%28Bollag+G[Author]%29+AND+Nature+Reviews+Drug+Discovery | issn = }}</ref>

=== Conserved Region 1 (CR1) ===

Conserved Region 1 [[Enzyme inhibitor|autoinhibits]] B-Raf's kinase domain (CR3) so that B-Raf signaling is regulated rather than constitutive.<ref name="Cutler Autoinhibition">{{cite journal | author = Cutler, RE et al. | title = Autoregulation of the Raf-1 serine/threonine kinase | journal = PNAS | volume = 95 | issue = 16 | pages = 9214–9219 | year = 1998 | month = August | pmid = 9689060 | doi = | url = http://www.pnas.org/content/95/16/9214 | issn = | pmc=21318}}</ref>  Residues 155-227<ref name="BRAF Uniprot">{{cite web | title = Serine/threonine protein kinase B-rAF | url = http://www.uniprot.org/uniprot/P15056 | accessdate = 4 Mar. 2013}}</ref> make up the [[Ras subfamily|Ras]]-binding domain (RBD), which binds to Ras-GTP's [[Effector (biology)|effector]] domain to release CR1 and halt kinase inhibition.  Residues 234-280 comprise a [[phorbol]] ester/[[diacylglycerol|DAG]]-binding [[zinc finger]] motif that participates in B-Raf membrane docking after Ras-binding.<ref name="BRAF Uniprot">{{cite web | title = Serine/threonine protein kinase B-rAF | url = http://www.uniprot.org/uniprot/P15056 | accessdate = 4 Mar. 2013}}</ref><ref name="pmid9069260">{{cite journal | author = Morrison DK, Cutler RE | title = The complexity of Raf-1 regulation | journal = Curr. Opin. Cell Biol. | volume = 9 | issue = 2 | pages = 174–179 | year = 1997 | month = April | pmid = 9069260 | doi = 10.1016/S0955-0674(97)80060-9 | url = http://dx.doi.org/10.1016/S0955-0674(97)80060-9 | issn = }}</ref>

=== Conserved Region 2 (CR2) ===

Conserved Region 2 (CR2) provides a flexible linker that connects CR1 and CR3 and acts as a hinge.

=== Conserved Region 3 (CR3) ===

[[File:BRAF Kinase Inactive.png|thumb | upright=1.5|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,<ref name="BRAF Uniprot">{{cite web | title = Serine/threonine protein kinase B-rAF | url = http://www.uniprot.org/uniprot/P15056 | accessdate = 4 Mar. 2013}}</ref> makes up B-Raf's enzymatic kinase domain.  This largely conserved structure<ref name="pmid7768349">{{cite journal | author = Hanks SK, Hunter T | title = Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification | journal = FASEB J. | volume = 9 | issue = 8 | pages = 576–96 | year = 1995 | month = May | pmid = 7768349 | doi = | url = http://www.fasebj.org/cgi/pmidlookup?view=long&pmid=7768349 | issn = }}</ref> is bi-lobal, connected by a short hinge region.<ref name="pmid15035987">{{cite journal | author = Wan, PT et al. | title = Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF | journal = Cell | volume = 116 | issue = 6 | pages = 855–67 | year = 2004 | month = March | pmid = 15035987 | doi = 10.1016/S0092-8674(04)00215-6| url = http://www.ncbi.nlm.nih.gov/pubmed?term=%28Wan%20PT[Author]%29%20AND%20Marais%20R[Author | issn = }}</ref> The smaller [[N-terminus|N]]-lobe (residues 457-530) is primarily responsible for [[adenosine triphosphate|ATP]] binding while the larger [[C-terminus|C]]-lobe (residues 535-717) binds [[Enzyme substrate (biology)|substrate]] proteins.<ref name="pmid7768349">{{cite journal | author = Hanks SK, Hunter T | title = Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification | journal = FASEB J. | volume = 9 | issue = 8 | pages = 576–96 | year = 1995 | month = May | pmid = 7768349 | doi = | url = http://www.fasebj.org/cgi/pmidlookup?view=long&pmid=7768349 | issn = }}</ref>  The active site is the cleft between the two lobes, and the catalytic [[Aspartic acid|Asp]]576 residue is located on the C-lobe, facing the inside of this cleft.<ref name="BRAF Uniprot">{{cite web | title = Serine/threonine protein kinase B-rAF | url = http://www.uniprot.org/uniprot/P15056 | accessdate = 4 Mar. 2013}}</ref><ref name="pmid7768349">{{cite journal | author = Hanks SK, Hunter T | title = Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification | journal = FASEB J. | volume = 9 | issue = 8 | pages = 576–96 | year = 1995 | month = May | pmid = 7768349 | doi = | url = http://www.fasebj.org/cgi/pmidlookup?view=long&pmid=7768349 | issn = }}</ref>

==== Important Subregions ====

'''P-Loop'''

The [[Walker motifs|P-loop]] of B-Raf (residues 464-471) stabilizes the non-transferable [[phosphate]] groups of ATP during enzyme ATP-binding.  Specifically, [[serine|S]]467, [[phenylalanine|F]]468, and [[glycine|G]]469 backbone [[amide]]s hydrogen-bond to the β-phosphate  of ATP to anchor the molecule.  B-Raf functional motifs have been determined by analyzing the homology of [[Protein kinase A|PKA]]  analyzed by Hanks and Hunter to the B-Raf kinase domain.<ref name="pmid7768349">{{cite journal | author = Hanks SK, Hunter T | title = Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification | journal = FASEB J. | volume = 9 | issue = 8 | pages = 576–96 | year = 1995 | month = May | pmid = 7768349 | doi = | url = http://www.fasebj.org/cgi/pmidlookup?view=long&pmid=7768349 | issn = }}</ref>

'''Nucleotide-Binding Pocket'''

[[valine|V]]471, [[cysteine|C]]532, [[tryptophan|W]]531, [[threonine|T]]529, [[leucine|L]]514, and [[alanine|A]]481 form a hydrophobic pocket within which the adenine of ATP is anchored through Van der Waals attractions upon ATP binding.<ref name="pmid7768349">{{cite journal | author = Hanks SK, Hunter T | title = Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification | journal = FASEB J. | volume = 9 | issue = 8 | pages = 576–96 | year = 1995 | month = May | pmid = 7768349 | doi = | url = http://www.fasebj.org/cgi/pmidlookup?view=long&pmid=7768349 | issn = }}</ref><ref name="pmid18287029">{{cite journal | author = Tsai J et al. | title = Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 105 | issue = 8 | pages = 3041–6 | year = 2008 | month = February | pmid = 18287029 | pmc = 2268581 | doi = 10.1073/pnas.0711741105 | url = | issn = }}</ref>

'''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, [[aspartic acid|D]]576 acts as a [[Base (chemistry)|proton acceptor]] to activate the [[nucleophile|nucleophilic]] hydroxyl oxygen on substrate serine or threonine residues, allowing the phosphate transfer reaction to occur mediated by [[acid catalysis|base-catalysis]].<ref name="pmid7768349">{{cite journal | author = Hanks SK, Hunter T | title = Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification | journal = FASEB J. | volume = 9 | issue = 8 | pages = 576–96 | year = 1995 | month = May | pmid = 7768349 | doi = | url = http://www.fasebj.org/cgi/pmidlookup?view=long&pmid=7768349 | issn = }}</ref>

'''DFG Motif'''

D594, F595, and G596 compose a motif central to B-Raf's function in both its inactive and active state.  In the inactive state, F595 occupies the nucleotide-binding pocket, prohibiting ATP from entering and decreasing the likelihood of enzyme catalysis.<ref name ="pmid23060265">{{cite journal | author = Bollag, G et al. | title = Vemurafenib: the first drug approved for BRAF-mutant cancer | journal = Nature Rev. Drug Discov. | volume = 11 | issue = 11 | pages = 873–886 | year = 2012 | month = November | pmid = 23060265| doi = 10.1038/nrd3847 | url = http://www.ncbi.nlm.nih.gov/pubmed/?term=%28Bollag+G[Author]%29+AND+Nature+Reviews+Drug+Discovery | issn = }}</ref><ref name="pmid18287029">{{cite journal | author = Tsai J et al. | title = Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 105 | issue = 8 | pages = 3041–6 | year = 2008 | month = February | pmid = 18287029 | pmc = 2268581 | doi = 10.1073/pnas.0711741105 | url = | issn = }}</ref><ref name="pmid20823850">{{cite journal | author = Bollag G et al. | title = Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma | journal = Nature | volume = 467 | issue = 7315 | pages = 596–599 | year = 2010 | month = September | pmid = 20823850 | pmc = 2948082 | doi = 10.1038/nature09454 | url = http://www.ncbi.nlm.nih.gov/pubmed/20823850 | issn = }}</ref>  In the active state, D594 [[chelation|chelates]] the [[divalent]] [[magnesium]] [[cation]] that stabilizes the β- and γ-phosphate groups of ATP, orienting the γ-phosphate for transfer.<ref name="pmid7768349">{{cite journal | author = Hanks SK, Hunter T | title = Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification | journal = FASEB J. | volume = 9 | issue = 8 | pages = 576–96 | year = 1995 | month = May | pmid = 7768349 | doi = | url = http://www.fasebj.org/cgi/pmidlookup?view=long&pmid=7768349 | issn = }}</ref>

'''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.<ref name="pmid15035987">{{cite journal | author = Wan, PT et al. | title = Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF | journal = Cell | volume = 116 | issue = 6 | pages = 855–67 | year = 2004 | month = March | pmid = 15035987 | doi = 10.1016/S0092-8674(04)00215-6| url = http://www.ncbi.nlm.nih.gov/pubmed?term=%28Wan%20PT[Author]%29%20AND%20Marais%20R[Author | issn = }}</ref>

== Enzymology ==

B-Raf is a [[serine/threonine-specific protein kinase]].  As such, it catalyzes the phosphorylation of [[serine]] and [[threonine]] residues in a [[consensus sequence]] on target proteins by [[Adenosine triphosphate|ATP]], yielding [[Adenosine diphosphate|ADP]] and a phosphorylated protein as products.<ref name="pmid7768349">{{cite journal | author = Hanks SK, Hunter T | title = Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification | journal = FASEB J. | volume = 9 | issue = 8 | pages = 576–96 | year = 1995 | month = May | pmid = 7768349 | doi = | url = http://www.fasebj.org/cgi/pmidlookup?view=long&pmid=7768349 | issn = }}</ref>  Since it is a highly regulated [[signal transduction]] [[Protein kinase|kinase]], B-Raf must first bind [[Ras subfamily|Ras]]-[[Guanosine triphosphate|GTP]] before becoming active as an enzyme.<ref name="pmid9069260">{{cite journal | author = Morrison DK, Cutler RE | title = The complexity of Raf-1 regulation | journal = Curr. Opin. Cell Biol. | volume = 9 | issue = 2 | pages = 174–179 | year = 1997 | month = April | pmid = 9069260| doi = 10.1016/S0955-0674(97)80060-9 | url = http://dx.doi.org/10.1016/S0955-0674(97)80060-9 | issn = }}</ref>  Once B-Raf is activated, a conserved protein kinase catalytic core phosphorylates protein substrates by promoting the nucleophilic attack of the activated substrate serine or threonine [[hydroxyl]] oxygen atom on the [[Alpha and beta carbon|γ-phosphate]] group of ATP through [[bimolecular nucleophilic substitution]].<ref name="pmid7768349">{{cite journal | author = Hanks SK, Hunter T | title = Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification | journal = FASEB J. | volume = 9 | issue = 8 | pages = 576–96 | year = 1995 | month = May | pmid = 7768349 | doi = | url = http://www.fasebj.org/cgi/pmidlookup?view=long&pmid=7768349 | issn = }}</ref><ref name="pmid3291115">{{cite journal | author = Hanks SK, Quinn AM, and Hunter T | title = The protein kinase family: conserved features and deduced phylogeny of the catalytic domains | journal = Science | volume = 241 | issue = | pages = 42–52 | year = 1988 | month = July | pmid = 3291115| doi = 10.1126/science.3291115 | url =  | issn = }}</ref><ref>{{cite journal | author = Hanks SK | title = Eukaryotic protein kinases | journal = Curr. Opin. Struct. Biol. | volume = 1 | issue = | pages = 369–383 | year = 1991 | month = June | pmid = | doi = 10.1016/0959-440X(91)90035-R | url = http://dx.doi.org/10.1016/0959-440X(91)90035-R | issn = }}</ref><ref name="pmid1956325">{{cite journal | author = Hanks SK, Quinn AM | title = Protein kinase catalytic domain sequence database: Identification of conserved features of primary structure and classification of family members | journal = Methods Enzymol. | volume = 200 | issue = | pages = 38–62 | year = 1991 | month = | pmid = 1956325 | doi = 10.1016/0076-6879(91)00126-H | url = http://dx.doi.org/10.1016/0076-6879(91)00126-H | issn = }}</ref>

=== Activating the Catalytic Domain ===

==== Releasing the Autoinhibitory CR1 Domain ====

The kinase (CR3) domain of human [[Raf kinase]]s is inhibited by two mechanisms: [[enzyme induction and inhibition|autoinhibition]] by its own [[Regulatory enzymes|regulatory]] [[Ras subfamily|Ras]]-[[Guanosine triphosphate|GTP]]-binding CR1 domain and a lack of [[Posttranslational modification|post-translational]] phosphorylation of key serine and [[tyrosine]] residues (S338 and Y341 for c-Raf) in the CR2 hinge region.  During B-Raf activation, the protein's [[enzyme induction and inhibition|autoinhibitory]] CR1 domain first binds Ras-GTP's [[Effector (biology)|effector]] domain to the CR1 Ras-binding domain (RBD) to release the kinase CR3 domain like other members of the human [[Raf kinase| Raf kinase family]].  The CR1-Ras interaction is later strengthened through the binding of the [[cysteine]]-rich subdomain (CRD) of CR1 to Ras and [[Cell membrane|membrane]] [[phospholipid]]s.<ref name="Cutler Autoinhibition">{{cite journal | author = Cutler, RE et al. | title = Autoregulation of the Raf-1 serine/threonine kinase | journal = PNAS | volume = 95 | issue = 16 | pages = 9214–9219 | year = 1998 | month = August | pmid = 9689060 | doi = | url = http://www.pnas.org/content/95/16/9214 | issn = | pmc=21318}}</ref>  Unlike [[ARAF|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.<ref name="pmid10205168">{{cite journal | author = Mason, CS et al. | title = Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation | journal = EMBO J. | volume = 18 | issue = 8 | pages = 2137–2148 | year = 1999 | month = April | pmid = 10205168 | doi = 10.1093/emboj/18.8.2137 | url = http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1171298/ | issn = | pmc=1171298}}</ref>  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.

==== Transforming the Protein Kinase CR3 Domain into Active Conformation ====

After the autoinhibitory CR1 regulatory domain is released, B-Raf's CR3 [[Protein kinase|kinase]] domain must change to its [[Adenosine triphosphate|ATP]]-binding active [[Conformational isomerism|conformer]] before it can catalyze protein phosphorylation.  In the inactive conformation, F595 of the DFG motif blocks the [[hydrophobe|hydrophobic]] [[adenine]] binding pocket while 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 [[Beta sheet|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 [[alpha helix|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.<ref name="pmid15035987">{{cite journal | author = Wan, PT et al. | title = Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF | journal = Cell | volume = 116 | issue = 6 | pages = 855–67 | year = 2004 | month = March | pmid = 15035987 | doi = 10.1016/S0092-8674(04)00215-6| url = http://www.ncbi.nlm.nih.gov/pubmed?term=%28Wan%20PT[Author]%29%20AND%20Marais%20R[Author | issn = }}</ref>

=== Enzymatic Mechanism ===

[[File:B-Raf Phosphorylation Mechanism.png|thumb| upright=2 | 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 [[adenosine diphosphate|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.<ref name="pmid7768349">{{cite journal | author = Hanks SK, Hunter T | title = Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification | journal = FASEB J. | volume = 9 | issue = 8 | pages = 576–96 | year = 1995 | month = May | pmid = 7768349 | doi = | url = http://www.fasebj.org/cgi/pmidlookup?view=long&pmid=7768349 | issn = }}</ref>

==== ATP Binding ====

B-Raf binds ATP by anchoring the adenine nucleotide in a [[nonpolar]] pocket (yellow, Figure 1) and orienting the molecule through hydrogen-bonding and electrostatic interactions with phosphate groups.  In addition to the P-loop and DFG motif phosphate binding described above, K483 and E500 play key roles in stabilizing non-transferable phosphate groups.  The positive charge on the primary [[amine]] of K483 allows it to stabilize the negative charge on ATP α- and β-phosphate groups when ATP binds.  When ATP is not present, the negative charge of the E500 [[carboxyl]] group balances this charge.<ref name="pmid7768349">{{cite journal | author = Hanks SK, Hunter T | title = Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification | journal = FASEB J. | volume = 9 | issue = 8 | pages = 576–96 | year = 1995 | month = May | pmid = 7768349 | doi = | url = http://www.fasebj.org/cgi/pmidlookup?view=long&pmid=7768349 | issn = }}</ref><ref name="pmid15035987">{{cite journal | author = Wan, PT et al. | title = Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF | journal = Cell | volume = 116 | issue = 6 | pages = 855–67 | year = 2004 | month = March | pmid = 15035987 | doi = 10.1016/S0092-8674(04)00215-6| url = http://www.ncbi.nlm.nih.gov/pubmed?term=%28Wan%20PT[Author]%29%20AND%20Marais%20R[Author | issn = }}</ref>

==== Protein 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 won't experience as much electron-electron repulsion when attacking the phosphate.  After the phosphate group is transferred, ADP and the new phosphoprotein are released.<ref name="pmid7768349">{{cite journal | author = Hanks SK, Hunter T | title = Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification | journal = FASEB J. | volume = 9 | issue = 8 | pages = 576–96 | year = 1995 | month = May | pmid = 7768349 | doi = | url = http://www.fasebj.org/cgi/pmidlookup?view=long&pmid=7768349 | issn = }}</ref>

=== Inhibitors ===

Since constituitively 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.<ref name="pmid15035987">{{cite journal | author = Wan, PT et al. | title = Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF | journal = Cell | volume = 116 | issue = 6 | pages = 855–67 | year = 2004 | month = March | pmid = 15035987 | doi = 10.1016/S0092-8674(04)00215-6| url = http://www.ncbi.nlm.nih.gov/pubmed?term=%28Wan%20PT[Author]%29%20AND%20Marais%20R[Author | issn = }}</ref><ref name="pmid18287029">{{cite journal | author = Tsai J et al. | title = Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 105 | issue = 8 | pages = 3041–6 | year = 2008 | month = February | pmid = 18287029 | pmc = 2268581 | doi = 10.1073/pnas.0711741105 | url = | issn = }}</ref><ref name="pmid20823850">{{cite journal | author = Bollag G et al. | title = Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma | journal = Nature | volume = 467 | issue = 7315 | pages = 596–599 | year = 2010 | month = September | pmid = 20823850 | pmc = 2948082 | doi = 10.1038/nature09454 | url = http://www.ncbi.nlm.nih.gov/pubmed/20823850 | issn = }}</ref>

==== BAY43-9006 (Sorafenib) ====

[[File:Sorafenib BRAF Labeled.png| thumb | left | upright=1.5 | Figure 3: B-Raf kinase domain locked in the inactive conformation by bound BAY43-9006. Hydrophobic interactions anchor BAY43-9006 in the ATP binding site while urea group hydrogen-bonding traps D594 of the DFG motif.  The BAY43-9006 trifluoromethyl [[phenyl]] ring further prohibits DFG motif and activation loop movement to the active confermer via steric blockage.]]

BAY43-9006 ([[Sorafenib]], Nexavar)is a V600E [[mutant]] B-Raf and [[C-Raf]] inhibitor approved by the [[Food and Drug Administration|FDA]] for the treatment of primary [[hepatocellular carcinoma|liver]] and [[renal cell carcinoma|kidney]] cancer.  Bay43-9006 disables the B-Raf [[protein kinase|kinase]] domain by locking the enzyme in its inactive form.  The inhibitor accomplishes this by blocking the ATP binding pocket through high-[[Affinity (pharmacology)|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.<ref name="pmid15035987">{{cite journal | author = Wan, PT et al. | title = Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF | journal = Cell | volume = 116 | issue = 6 | pages = 855–67 | year = 2004 | month = March | pmid = 15035987 | doi = 10.1016/S0092-8674(04)00215-6| url = http://www.ncbi.nlm.nih.gov/pubmed?term=%28Wan%20PT[Author]%29%20AND%20Marais%20R[Author | issn = }}</ref>

The distal [[pyridine|pyridyl]] ring of BAY43-9006 anchors in the hydrophobic nucleotide-binding pocket of the kinase N-lobe, interacting with W531, F583, and F595.  The hydrophobic interactions with catalytic loop F583 and DFG motif F595 stabilize the inactive conformation of these structures, decreasing the likelihood of enzyme activation.  Further hydrophobic interaction of K483, L514, and T529 with the center phenyl ring increase the [[Affinity (pharmacology)|affinity]] of the kinase domain for the inhibitor.  Hydrophobic interaction of F595 with the center ring as well decreases the energetic favorability of a DFG conformation switch further.  Finally, polar interactions of BAY43-9006 with the kinase domain continue this trend of increasing enzyme affinity for the inhibitor and stabilizing DFG residues in the inactive conformation.  E501 and C532 hydrogen bond the [[urea]] and pyridyl groups of the inhibitor respectively while the [[urea]] [[carbonyl]] accepts a hydrogen bond from D594's backbone [[amide]] nitrogen to lock the DFG motif in place.<ref name="pmid15035987">{{cite journal | author = Wan, PT et al. | title = Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF | journal = Cell | volume = 116 | issue = 6 | pages = 855–67 | year = 2004 | month = March | pmid = 15035987 | doi = 10.1016/S0092-8674(04)00215-6| url = http://www.ncbi.nlm.nih.gov/pubmed?term=%28Wan%20PT[Author]%29%20AND%20Marais%20R[Author | issn = }}</ref>

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.<ref name="pmid15035987">{{cite journal | author = Wan, PT et al. | title = Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF | journal = Cell | volume = 116 | issue = 6 | pages = 855–67 | year = 2004 | month = March | pmid = 15035987 | doi = 10.1016/S0092-8674(04)00215-6| url = http://www.ncbi.nlm.nih.gov/pubmed?term=%28Wan%20PT[Author]%29%20AND%20Marais%20R[Author | issn = }}</ref>

==== PLX 4032 (Vemurafenib)====

[[File:Vemurafenib.png|thumb| upright=1.5 |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 [[Food and Drug Administration|FDA]] for the treatment of late-stage [[melanoma]].<ref name ="pmid23060265">{{cite journal | author = Bollag, G et al. | title = Vemurafenib: the first drug approved for BRAF-mutant cancer | journal = Nature Rev. Drug Discov. | volume = 11 | issue = 11 | pages = 873–886 | year = 2012 | month = November | pmid = 23060265 | doi = 10.1038/nrd3847 | url = http://www.ncbi.nlm.nih.gov/pubmed/?term=%28Bollag+G[Author]%29+AND+Nature+Reviews+Drug+Discovery | issn = }}</ref>  Unlike [[Sorafenib|BAY43-9006]], which inhibits the inactive form of the kinase domain, Vemurafenib inhibits the active "DFG-in" form of the kinase,<ref name="pmid18287029">{{cite journal | author = Tsai J et al. | title = Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 105 | issue = 8 | pages = 3041–6 | year = 2008 | month = February | pmid = 18287029 | pmc = 2268581 | doi = 10.1073/pnas.0711741105 | url = | issn = }}</ref><ref name="pmid20823850">{{cite journal | author = Bollag G et al. | title = Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma | journal = Nature | volume = 467 | issue = 7315 | pages = 596–599 | year = 2010 | month = September | pmid = 20823850 | pmc = 2948082 | doi = 10.1038/nature09454 | url = http://www.ncbi.nlm.nih.gov/pubmed/20823850 | issn = }}</ref> 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 [[phenyl]] ring added for [[Pharmacokinetics|pharmacokinetic]] reasons,<ref name="pmid20823850">{{cite journal | author = Bollag G et al. | title = Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma | journal = Nature | volume = 467 | issue = 7315 | pages = 596–599 | year = 2010 | month = September | pmid = 20823850 | pmc = 2948082 | doi = 10.1038/nature09454 | url = http://www.ncbi.nlm.nih.gov/pubmed/20823850 | issn = }}</ref> PLX4720's mode of action is equivalent to Vemurafenib's.  PLX4720 has good affinity for the ATP binding site partially because its anchor region, a 7-aza[[indole]] [[Bicyclic molecule|bicyclic]], only differs from the natural adenine that occupies the site in two places where nitrogen atoms have been replaced by carbon.  This enables strong intermolecular interactions like N7 hydrogen bonding to C532 and N1 hydrogen bonding to Q530 to be preserved.  Excellent fit within the ATP-binding hydrophobic pocket (C532, W531, T529, L514, A481) increases binding affinity as well.  [[Ketone]] linker hydrogen bonding to water and difluoro-phenyl fit in a second hydrophobic pocket (A481, V482, K483, V471, I527, T529, L514, and F583) contribute to the exceptionally high binding affinity overall.  Selective binding to active Raf is accomplished by the terminal propyl group that binds to a Raf-selective pocket created by a shift of the αC helix.  Selectivity for the active conformation of the kinase is further increased by a pH-sensitive deprotonated [[Sulfonamide (medicine)|sulfonamide]] group that is stabilized by hydrogen bonding with the backbone peptide NH of D594 in the active state.  In the inactive state, the inhibitor's sulfonamide group interacts with the backbone [[carbonyl]] of that residue instead, creating repulsion.  Thus, Vemurafenib binds preferentially to the active state of B-Raf's kinase domain.<ref name="pmid18287029">{{cite journal | author = Tsai J et al. | title = Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 105 | issue = 8 | pages = 3041–6 | year = 2008 | month = February | pmid = 18287029 | pmc = 2268581 | doi = 10.1073/pnas.0711741105 | url = | issn = }}</ref><ref name="pmid20823850">{{cite journal | author = Bollag G et al. | title = Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma | journal = Nature | volume = 467 | issue = 7315 | pages = 596–599 | year = 2010 | month = September | pmid = 20823850 | pmc = 2948082 | doi = 10.1038/nature09454 | url = http://www.ncbi.nlm.nih.gov/pubmed/20823850 | issn = }}</ref>

==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.<ref name="pmid16825433">{{cite journal | author = Roberts A, Allanson J, Jadico SK, Kavamura MI, Noonan J, Opitz JM, Young T, Neri G | title = The cardiofaciocutaneous syndrome | journal = J. Med. Genet. | volume = 43 | issue = 11 | pages = 833–42 | year = 2006 | month = November | pmid = 16825433 | pmc = 2563180 | doi = 10.1136/jmg.2006.042796 | url = | issn = }}</ref>

Acquired mutations in this gene have been found in cancers, including [[non-Hodgkin lymphoma]], [[colorectal cancer]], malignant [[melanoma]], [[Papillary thyroid cancer|papillary thyroid carcinoma]], [[non-small-cell lung carcinoma]], and [[adenocarcinoma of the lung]].<ref name="entrez"/>

The V600E mutation of the BRAF gene has been associated with [[hairy cell leukemia]] in numerous [http://www.ncbi.nlm.nih.gov/pubmed/22246856 studies] and has been suggested for use in screening for [[Lynch syndrome]] to reduce the number of patients undergoing unnecessary [[MLH1]] sequencing.<ref name="pmid19125127">{{cite journal | author = Palomaki GE, McClain MR, Melillo S, Hampel HL, Thibodeau SN | title = EGAPP supplementary evidence review: DNA testing strategies aimed at reducing morbidity and mortality from Lynch syndrome | journal = Genet. Med. | volume = 11 | issue = 1 | pages = 42–65 | year = 2009 | month = January | pmid = 19125127 | doi = 10.1097/GIM.0b013e31818fa2db  }}</ref>

===BRAF 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 [[melanoma]]s and [[nevus|nevi]], to as little as 0-18% in other [[tumor]]s, such as 1-3% in lung cancers and 5% in [[colorectal cancer]].<ref name="pmid12970315">{{cite journal | author = Namba H, Nakashima M, Hayashi T, Hayashida N, Maeda S, Rogounovitch TI, Ohtsuru A, Saenko VA, Kanematsu T, Yamashita S | title = Clinical implication of hot spot BRAF mutation, V599E, in papillary thyroid cancers | journal = J. Clin. Endocrinol. Metab. | volume = 88 | issue = 9 | pages = 4393–7 | year = 2003 | month = September | pmid = 12970315 | doi = 10.1210/jc.2003-030305| url = http://jcem.endojournals.org/cgi/content/abstract/88/9/4393 | issn = }}</ref>  In 90% of the cases, thymine is substituted with adenine at nucleotide 1799. This leads to valine (V) being substituted for by glutamate (E) at codon 600 (now referred to as V600E) in the activation segment that has been found in human cancers.<ref name="pmid18428050">{{cite journal | author = Tan YH, Liu Y, Eu KW, Ang PW, Li WQ, Salto-Tellez M, Iacopetta B, Soong R | title = Detection of BRAF V600E mutation by pyrosequencing | journal = Pathology | volume = 40 | issue = 3 | pages = 295–8 | year = 2008 | month = April | pmid = 18428050 | doi = 10.1080/00313020801911512 }}</ref> This mutation has been widely observed in [[papillary thyroid carcinoma]], colorectal cancer, melanoma and [[non-small-cell lung cancer]].<ref name="pmid16403224">{{cite journal | author = Li WQ, Kawakami K, Ruszkiewicz A, Bennett G, Moore J, Iacopetta B | title = BRAF mutations are associated with distinctive clinical, pathological and molecular features of colorectal cancer independently of microsatellite instability status | journal = Mol. Cancer | volume = 5 | issue = 1| pages = 2 | year = 2006 | pmid = 16403224 | pmc = 1360090 | doi = 10.1186/1476-4598-5-2 }}</ref><ref name="pmid17065421">{{cite journal | author = Benlloch S, Payá A, Alenda C, Bessa X, Andreu M, Jover R, Castells A, Llor X, Aranda FI, Massutí B | title = Detection of BRAF V600E mutation in colorectal cancer: comparison of automatic sequencing and real-time chemistry methodology | journal = J Mol Diagn | volume = 8 | issue = 5 | pages = 540–3 | year = 2006 | month = November | pmid = 17065421 | pmc = 1876165 | doi = 10.2353/jmoldx.2006.060070 }}</ref><ref name="pmid14734469">{{cite journal | author = Deng G, Bell I, Crawley S, Gum J, Terdiman JP, Allen BA, Truta B, Sleisenger MH, Kim YS | title = BRAF mutation is frequently present in sporadic colorectal cancer with methylated hMLH1, but not in hereditary nonpolyposis colorectal cancer | journal = Clin. Cancer Res. | volume = 10 | issue = 1 Pt 1 | pages = 191–5 | year = 2004 | month = January | pmid = 14734469 | doi = 10.1158/1078-0432.CCR-1118-3 }}</ref><ref name="pmid15277467">{{cite journal | author = Gear H, Williams H, Kemp EG, Roberts F | title = BRAF mutations in conjunctival melanoma | journal = Invest. Ophthalmol. Vis. Sci. | volume = 45 | issue = 8 | pages = 2484–8 | year = 2004 | month = August | pmid = 15277467 | doi = 10.1167/iovs.04-0093 }}</ref><ref name="pmid14679157">{{cite journal | author = Maldonado JL, Fridlyand J, Patel H, Jain AN, Busam K, Kageshita T, Ono T, Albertson DG, Pinkel D, Bastian BC | title = Determinants of BRAF mutations in primary melanomas | journal = J. Natl. Cancer Inst. | volume = 95 | issue = 24 | pages = 1878–90 | year = 2003 | month = December | pmid = 14679157 | doi = 10.1093/jnci/djg123 }}</ref><ref name="pmid15126572">{{cite journal | author = Puxeddu E, Moretti S, Elisei R, Romei C, Pascucci R, Martinelli M, Marino C, Avenia N, Rossi ED, Fadda G, Cavaliere A, Ribacchi R, Falorni A, Pontecorvi A, Pacini F, Pinchera A, Santeusanio F | title = BRAF(V599E) mutation is the leading genetic event in adult sporadic papillary thyroid carcinomas | journal = J. Clin. Endocrinol. Metab. | volume = 89 | issue = 5 | pages = 2414–20 | year = 2004 | month = May | pmid = 15126572 | doi = 10.1210/jc.2003-031425  }}</ref><ref name="pmid18682506">{{cite journal | author = Elisei R, Ugolini C, Viola D, Lupi C, Biagini A, Giannini R, Romei C, Miccoli P, Pinchera A, Basolo F | title = BRAF(V600E) mutation and outcome of patients with papillary thyroid carcinoma: a 15-year median follow-up study | journal = J. Clin. Endocrinol. Metab. | volume = 93 | issue = 10 | pages = 3943–9 | year = 2008 | month = October | pmid = 18682506 | doi = 10.1210/jc.2008-0607  }}</ref> In 2010 a team of scientists demonstrated presence of BRAF-V600E mutation in 57% of Langerhans cell histiocytosis patients.<ref name="pmid22017623">{{cite journal | author = Badalian-Very G, Vergilio JA, Degar BA, Rodriguez-Galindo C, Rollins BJ | title = Recent advances in the understanding of Langerhans cell histiocytosis | journal = Br. J. Haematol. | volume = 156 | issue = 2 | pages = 163–72 | year = 2012 | month = January | pmid = 22017623 | doi = 10.1111/j.1365-2141.2011.08915.x  }}</ref> A team of Italian scientists used massively parallel sequencing to pinpoint mutation V600E as a likely driver mutation in 100% of cases of [[hairy cell leukaemia]].<ref name="TiacciTrifonov2011">{{cite journal | author = Tiacci E, Trifonov V, Schiavoni G, Holmes A, Kern W, Martelli MP, Pucciarini A, Bigerna B, Pacini R, Wells VA, Sportoletti P, Pettirossi V, Mannucci R, Elliott O, Liso A, Ambrosetti A, Pulsoni A, Forconi F, Trentin L, Semenzato G, Inghirami G, Capponi M, Di Raimondo F, Patti C, Arcaini L, Musto P, Pileri S, Haferlach C, Schnittger S, Pizzolo G, Foà R, Farinelli L, Haferlach T, Pasqualucci L, Rabadan R, Falini B | title = BRAF mutations in hairy-cell leukemia | journal = N. Engl. J. Med. | volume = 364 | issue = 24 | pages = 2305–15 | year = 2011 | month = June | pmid = 21663470 | doi = 10.1056/NEJMoa1014209 | laysummary = http://scienceblog.cancerresearchuk.org/2011/06/11/research-on-hairy-cell-leukaemia-shows-the-promise-of-new-dna-scanning-technologies/ | laysource = Science Update blog: Cancer Research UK }}</ref>

Other mutations which have been found are R461I, I462S, G463E, G463V, G465A, G465E, G465V, G468A, G468E, 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.<ref name="pmid15035987">{{cite journal | author = Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, Jones CM, Marshall CJ, Springer CJ, Barford D, Marais R | title = Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-Raf | journal = Cell | volume = 116 | issue = 6 | pages = 855–67 | year = 2004 | month = March | pmid = 15035987 | doi = 10.1016/S0092-8674(04)00215-6 }}</ref> 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 [[Mitogen-activated protein kinase kinase|MEK]] may also vary. In the same paper it has been reported that 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 [[Extracellular signal-regulated kinases|ERK]].

===B-Raf inhibitors in the clinic=== <!-- redirect target for Braf inhibitor -->
As mentioned above, some pharmaceutical firms are developing specific inhibitors of mutated B-raf protein for [[anticarcinogen|anticancer]] use because B-Raf is a well-understood, high yield target.<ref name="pmid18287029">{{cite journal | author = Tsai J, Lee JT, Wang W, Zhang J, Cho H, Mamo S, Bremer R, Gillette S, Kong J, Haass NK, Sproesser K, Li L, Smalley KS, Fong D, Zhu YL, Marimuthu A, Nguyen H, Lam B, Liu J, Cheung I, Rice J, Suzuki Y, Luu C, Settachatgul C, Shellooe R, Cantwell J, Kim SH, Schlessinger J, Zhang KY, West BL, Powell B, Habets G, Zhang C, Ibrahim PN, Hirth P, Artis DR, Herlyn M, Bollag G | title = Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 105 | issue = 8 | pages = 3041–6 | year = 2008 | month = February | pmid = 18287029 | pmc = 2268581 | doi = 10.1073/pnas.0711741105 | url = | issn = }}</ref><ref name="pmid17145850">{{cite journal | author = King AJ, Patrick DR, Batorsky RS, Ho ML, Do HT, Zhang SY, Kumar R, Rusnak DW, Takle AK, Wilson DM, Hugger E, Wang L, Karreth F, Lougheed JC, Lee J, Chau D, Stout TJ, May EW, Rominger CM, Schaber MD, Luo L, Lakdawala AS, Adams JL, Contractor RG, Smalley KS, Herlyn M, Morrissey MM, Tuveson DA, Huang PS | title = Demonstration of a genetic therapeutic index for tumors expressing oncogenic BRAF by the kinase inhibitor SB-590885 | journal = Cancer Res. | volume = 66 | issue = 23 | pages = 11100–5 | year = 2006 | month = December | pmid = 17145850 | doi = 10.1158/0008-5472.CAN-06-2554 | url = | issn = }}</ref>  [[Vemurafenib]] (RG7204 or PLX4032), licensed by the US [[Food and Drug Administration]] as Zelboraf for the treatment of metastatic melanoma in August 2011, is the current state-of-the-art example for why active B-Raf inhibitors are being pursued as drug candidates.  Vemurafenib is biochemically interesting as a mechanism to target cancer due to its high efficacy and selectivity.<ref name ="pmid23060265">{{cite journal | author = Bollag, G et al. | title = Vemurafenib: the first drug approved for BRAF-mutant cancer | journal = Nature Rev. Drug Discov. | volume = 11 | issue = 11 | pages = 873–886 | year = 2012 | month = November | pmid = 23060265 | doi = 10.1038/nrd3847 | url = http://www.ncbi.nlm.nih.gov/pubmed/?term=%28Bollag+G[Author]%29+AND+Nature+Reviews+Drug+Discovery | issn = }}</ref>  In Phase II<ref name ="pmid22356324">{{cite journal | author = Sosman, JA et al. | title = Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib | journal = N Engl J Med. | volume = 366 | issue = 8 | pages = 707–714 | year = 2012 | month = February | pmid = 22356324 | doi = 10.1056/NEJMoa1112302 | url = | issn = }}</ref> and Phase III<ref name ="pmid21639808">{{cite journal | author = Chapman, PB et al. | title = Improved survival with vemurafenib in melanoma with BRAF V600E mutation | journal = N Engl J Med. | volume = 364 | issue = 26 | pages = 2507–16 | year = 2011 | month = June | pmid = 21639808 | doi = 10.1056/NEJMoa1103782 | url = | issn = }}</ref> clinical trials, B-Raf not only increased metastatic melanoma patient chance of survival but raised the response rate to treatment from 7-12% to 53% in the same amount of time compared to the former best chemotherapeutic treatment: dacarbazine.  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.<ref name ="pmid23302800">{{cite journal | author = Das Thakur et al. | title = Modelling vemurafenib resistance in melanoma
reveals a strategy to forestall drug resistance | journal = Nature | volume = 494 | issue = | pages = 251–256 | year = 2013 | month = February | pmid = 23302800 | doi = 10.1038/nature11814 | url = | issn = }}</ref>  While the mechanisms of this resistance are still disputed, some hypotheses include the overexpression of B-Raf to compensate for high concentrations of Vemurafenib<ref name ="pmid23302800">{{cite journal | author = Das Thakur et al. | title = Modelling vemurafenib resistance in melanoma
reveals a strategy to forestall drug resistance | journal = Nature | volume = 494 | issue = | pages = 251–255 | year = 2013 | month = February | pmid = 23302800 | doi = 10.1038/nature11814 | url = | issn = }}</ref> and upstream upregulation of growth signaling.<ref name ="pmid21107323">{{cite journal | author = Nazarian et al. | title = Melanomas acquire resistance to B-RAF(V600E)
inhibition by RTK or N-RAS upregulation | journal = Nature | volume = 468 | issue = | pages = 973–977 | year = 2010 | month = December | pmid = 21107323 | doi = 10.1038/nature09626 | url = | issn = }}</ref>

More general B-[[raf inhibitor]]s include [[GDC-0879]], [[PLX-4720]], [[Sorafenib]] Tosylate. [[dabrafenib]], [[LGX818]]

==Interactions==
BRAF (gene) has been shown to [[Protein-protein interaction|interact]] with [[YWHAB]],<ref name="pmid17353931">{{cite journal | author = Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T, Figeys D | title = Large-scale mapping of human protein-protein interactions by mass spectrometry | journal = Mol. Syst. Biol. | volume = 3 | issue = 1| pages = 89 | year = 2007 | pmid = 17353931 | pmc = 1847948 | doi = 10.1038/msb4100134 | url = | issn = }}</ref><ref name="pmid10931830">{{cite journal | author = Qiu W, Zhuang S, von Lintig FC, Boss GR, Pilz RB | title = Cell type-specific regulation of B-Raf kinase by cAMP and 14-3-3 proteins | journal = J. Biol. Chem. | volume = 275 | issue = 41 | pages = 31921–9 | year = 2000 | month = October | pmid = 10931830 | doi = 10.1074/jbc.M003327200 | url = | issn = }}</ref> [[C-Raf]],<ref name="pmid11325826">{{cite journal | author = Weber CK, Slupsky JR, Kalmes HA, Rapp UR | title = Active Ras induces heterodimerization of cRaf and BRaf | journal = Cancer Res. | volume = 61 | issue = 9 | pages = 3595–8 | year = 2001 | month = May | pmid = 11325826 | doi = | url = | issn = }}</ref> [[AKT1]]<ref name="pmid10869359">{{cite journal | author = Guan KL, Figueroa C, Brtva TR, Zhu T, Taylor J, Barber TD, Vojtek AB | title = Negative regulation of the serine/threonine kinase B-Raf by Akt | journal = J. Biol. Chem. | volume = 275 | issue = 35 | pages = 27354–9 | year = 2000 | month = September | pmid = 10869359 | doi = 10.1074/jbc.M004371200 | url = | issn = }}</ref> and [[HRAS]].<ref name="pmid9154803">{{cite journal | author = Stang S, Bottorff D, Stone JC | title = Interaction of activated Ras with Raf-1 alone may be sufficient for transformation of rat2 cells | journal = Mol. Cell. Biol. | volume = 17 | issue = 6 | pages = 3047–55 | year = 1997 | month = June | pmid = 9154803 | pmc = 232157 | doi = | url = | issn = }}</ref><ref name="pmid7706312">{{cite journal | author = Reuter CW, Catling AD, Jelinek T, Weber MJ | title = Biochemical analysis of MEK activation in NIH3T3 fibroblasts. Identification of B-Raf and other activators | journal = J. Biol. Chem. | volume = 270 | issue = 13 | pages = 7644–55 | year = 1995 | month = March | pmid = 7706312 | doi = 10.1074/jbc.270.13.7644| url = | issn = }}</ref>

==References==
{{Reflist|2}}

==Further reading==
{{Refbegin|colwidth=35em}}
*{{cite journal |author=Sithanandam G, Kolch W, Duh FM, Rapp UR |title=Complete coding sequence of a human B-raf cDNA and detection of B-raf protein kinase with isozyme specific antibodies |journal=[[Oncogene (journal)|Oncogene]] |volume=5 |issue=12 |pages=1775–80 |year=1991 |pmid=2284096 |doi=  }} 
*{{cite journal |author=Garnett MJ, Marais R |title=Guilty as charged: B-Raf is a human oncogene |journal=[[Cancer Cell]] |volume=6 |issue=4 |pages=313–9 |year=2004 |pmid=15488754 |doi=10.1016/j.ccr.2004.09.022 }}
*{{cite journal |author=Quiros RM, Ding HG, Gattuso P, ''et al.'' |title=Evidence that one subset of anaplastic thyroid carcinomas are derived from papillary carcinomas due to BRAF and p53 mutations |journal=[[Cancer (journal)|Cancer]] |volume=103 |issue=11 |pages=2261–8 |year=2005 |pmid=15880523 |doi=10.1002/cncr.21073 }}
*{{cite journal |author=Karbowniczek M, Henske EP |title=The role of tuberin in cellular differentiation: are B-Raf and MAPK involved? |journal=[[Ann N Y Acad Sci]] |volume=1059 |issue= 1|pages=168–73 |year=2006 |pmid=16382052 |doi=10.1196/annals.1339.045 }}
*{{cite journal |author=Ciampi R, Nikiforov YE |title=RET/PTC rearrangements and BRAF mutations in thyroid tumorigenesis |journal=[[Endocrinology (journal)|Endocrinology]]|volume=148 |issue= 3 |pages=936–41 |year=2007 |pmid=16946010 |doi=10.1210/en.2006-0921 }} *{{cite journal |author=Espinosa AV, Porchia L, Ringel MD |title=Targeting BRAF in thyroid cancer |journal=[[British Journal of Cancer]] |volume=96 |issue=1 |pages=16–20 |year=2007 |pmid=17179987 |doi=10.1038/sj.bjc.6603520 |pmc=2360215 }}
{{Refend}}

==External links==
* [http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=noonan  GeneReviews/NCBI/NIH/UW entry on Noonan syndrome]
* [http://www.ncbi.nlm.nih.gov/books/NBK1186/  GeneReviews/NCBI/NIH/UW entry on Cardiofaciocutaneous Syndrome]
* [http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=leopard  GeneReviews/NCBI/NIH/UW entry on LEOPARD Syndrome]
* {{cite web |url = http://www.cancer.gov/dictionary?CdrID=561325  |title = BRAF gene |publisher = NCI Dictionary of Cancer Terms |accessdate=2007-11-25}}
* [http://scienceblog.cancerresearchuk.org/2009/08/24/high-impact-science-%E2%80%93-finding-faults-in-braf/ Finding faults in BRAF] - Cancer Research UK blog post about the discovery of cancer-causing BRAF mutations (incl video)

{{Serine/threonine-specific protein kinases}}

[[Category:Oncogenes]]