RAF activation

Stable Identifier
Homo sapiens
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Mammals have three RAF isoforms, A, B and C, that are activated downstream of RAS and stimulate the MAPK pathway. Although CRAF (also known as RAF-1) was the first identified and remains perhaps the best studied, BRAF is most similar to the RAF expressed in other organisms. Notably, MAPK (ERK) activation is more compromised in BRAF-deficient cells than in CRAF or ARAF deficient cells (Bonner et al, 1985; Mikula et al, 2001, Huser et al, 2001, Mercer et al, 2002; reviewed in Leicht et al, 2007; Matallanas et al, 2011; Cseh et al, 2014). Consistent with its important role in MAPK pathway activation, mutations in the BRAF gene, but not in those for A- or CRAF, are associated with cancer development (Davies et al, 2002; reviewed in Leicht et al, 2007). ARAF and CRAF may have arisen through gene duplication events, and may play additional roles in MAPK-independent signaling (Hindley and Kolch, 2002; Murakami and Morrison, 2001).
Despite divergences in function, all mammalian RAF proteins share three conserved regions (CRs) and each interacts with RAS and MEK proteins, although with different affinities. The N-terminal CR1 contains a RAS-binding domain (RBD) and a cysteine-rich domain (CRD) that mediate interactions with RAS and the phospholipid membrane. CR2 contains inhibitory phosphorylation sites that impact RAS binding and RAF activation, while the C-terminal CR3 contains the bi-lobed kinase domain with its activation loop, and an adjacent upstream "N-terminal acidic motif" -S(S/G)YY in C- and A-RAF,respectively, and SSDD in B-RAF - that is required for RAF activation (Tran et al, 2005; Dhillon et al, 2002; Chong et al, 2001; Cutler et al, 1998; Chong et al, 2003; reviewed in Matallanas et al, 2011).

Regulation of RAF activity involves multiple phosphorylation and dephosphorylation events, intramolecular conformational changes, homo- and heterodimerization between RAF monomers and changes to protein binding partners, including scaffolding proteins which bring pathway members together (reviewed in Matallanas et al, 2011; Cseh et al, 2014). The details of this regulation are not completely known and differ slightly from one RAF isoform to another. Briefly, in the inactive state, RAF phosphorylation on conserved serine residues in CR2 promote an interaction with 14-3-3 dimers, maintaining the kinase in a closed conformation. Upon RAS activation, these sites are dephosphorylated, allowing the RAF CRD and RBD to bind RAS and phospholipids, facilitating membrane recruitment. RAF activation requires homo- or heterodimerization, which promotes autophosphorylation in the activation loop of the receiving monomer. Of the three isoforms, only BRAF is able to initiate this allosteric activation of other RAF monomers (Hu et al, 2013; Heidorn et al, 2010; Garnett et al, 2005). This activity depends on negative charge in the N-terminal acidic region (NtA; S(S/G)YY or SSDD) adjacent to the kinase domain. In BRAF, this region carries permanent negative charge due to the presence of the two aspartate residues in place of the tyrosine residues of A- and CRAF. In addition, unique to BRAF, one of the serine residues of the NtA is constitutively phosphorylated. In A- and CRAF, residues in this region are subject to phosphorylation by activated MEK downstream of RAF activation, establishing a positive feedback loop and allowing activated A- and CRAF monomers to act as transactivators in turn (Hu et al, 2013; reviewed in Cseh et al, 2014). RAF signaling is terminated through dephosphorylation of the NtA region and phosphorylation of the residues that mediate the inhibitory interaction with 14-3-3, promoting a return to the inactive state (reviewed in Matallanas et al, 2011; Cseh et al, 2014).

Literature References
PubMed ID Title Journal Year
21779496 Raf family kinases: old dogs have learned new tricks

Romano, D, Matallanas, D, Rauch, J, Zebisch, A, Birtwistle, M, Kolch, W, von Kriegsheim, A

Genes Cancer 2011
11782426 Regulation of Raf-1 activation and signalling by dephosphorylation

Meikle, S, Yazici, Z, Eulitz, M, Kolch, W, Dhillon, AS

EMBO J. 2002
11447113 Positive and negative regulation of Raf kinase activity and function by phosphorylation

Guan, KL, Lee, J, Chong, H

EMBO J. 2001
11821947 ERK signalling and oncogene transformation are not impaired in cells lacking A-Raf

Pritchard, C, Kiernan, M, Mercer, K, Hüser, M, Chiloeches, A, Marais, R

Oncogene 2002
24937142 "RAF" neighborhood: protein-protein interaction in the Raf/Mek/Erk pathway

Baccarini, M, Cseh, B, Doma, E

FEBS Lett. 2014
12865432 Regulation of Raf through phosphorylation and N terminus-C terminus interaction

Guan, KL, Chong, H

J. Biol. Chem. 2003
2993863 Structure and biological activity of human homologs of the raf/mil oncogene

Bonner, TI, Mark, G, Gunnell, MA, Sutrave, P, Kerby, SB, Rapp, UR

Mol. Cell. Biol. 1985
11950876 Extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK)-independent functions of Raf kinases

Hindley, A, Kolch, W

J. Cell. Sci. 2002
23993095 Allosteric activation of functionally asymmetric RAF kinase dimers

Stites, EC, Taylor, SS, Yu, H, Hu, J, Germino, EA, Meharena, HS, Stork, PJ, Kornev, AP, Shaw, AS

Cell 2013
11296228 Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene

Mikula, M, Schreiber, M, Husak, Z, Kucerova, L, Baccarini, M, Wagner, EF, Zatloukal, K, Wieser, R, Beug, H, Rüth, J

EMBO J. 2001
11579234 Raf-1 without MEK?

Morrison, DK, Murakami, MS

Sci. STKE 2001
9689060 Autoregulation of the Raf-1 serine/threonine kinase

Stephens, RM, Morrison, DK, Cutler, RE, Saracino, MR

Proc. Natl. Acad. Sci. U.S.A. 1998
20141835 Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF

Reis-Filho, JS, Heidorn, SJ, Springer, CJ, Pritchard, C, Nourry, A, Milagre, C, Whittaker, S, Dhomen, N, Marais, R, Niculescu-Duvas, I, Hussain, J

Cell 2010
16364920 Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimerization

Garnett, MJ, Barford, D, Paterson, H, Rana, S, Marais, R

Mol. Cell 2005
17555829 Raf kinases: function, regulation and role in human cancer

Dobson, M, Kaplun, L, Singh-Gupta, V, Balan, V, Tzivion, G, Leicht, DT, Kaplun, A

Biochim. Biophys. Acta 2007
12068308 Mutations of the BRAF gene in human cancer

Maitland, N, Jayatilake, H, Futreal, PA, Yuen, ST, Marshall, CJ, Menzies, A, Parker, A, Chenevix-Trench, G, Leung, SY, Garnett, MJ, Davis, N, Shipley, J, Cooper, C, Darrow, TL, Marais, R, Paterson, H, Palmieri, G, Floyd, Y, Bottomley, W, Bigner, DD, Bignell, GR, Stevens, C, Cossu, A, Hall, S, Ho, JW, Wilson, R, Mould, C, Gusterson, BA, Watt, S, Cox, C, Riggins, GJ, Flanagan, A, Gray, K, Wooster, R, Hooper, S, Teague, J, Ewing, R, Edkins, S, Seigler, HF, Hargrave, D, Hawes, R, Kosmidou, V, Nicholson, A, Stephens, P, Weber, BL, Hughes, J, Davies, H, Woffendin, H, Stratton, MR, Clegg, S, Dicks, E, Pritchard-Jones, K

Nature 2002
11296227 MEK kinase activity is not necessary for Raf-1 function

Pritchard, C, Luckett, J, Giblett, S, Iwobi, M, Brown, J, Mercer, K, Hüser, M, Sun, XM, Chiloeches, A, Marais, R

EMBO J. 2001
15710605 B-Raf and Raf-1 are regulated by distinct autoregulatory mechanisms

Frost, JA, Wu, X, Tran, NH

J. Biol. Chem. 2005
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