Cell Vol 116 855 867 March 19 2004 Copyright 2004 by Cell Press Mechanism of Activation of the RAF ERK Signaling Pathway by Oncogenic Mutations of B RAF Paul T C Wan 1 6 Mathew J Garnett 2 6 S Mark Roe 1 Sharlene Lee 3 Dan Niculescu Duvaz 4 Valerie M Good 1 Cancer Genome Project 5 C Michael Jones 3 Christopher J Marshall 2 Caroline J Springer 4 David Barford 1 and Richard Marais2 1 Section of Structural Biology 2 Cancer Research UK Centre for Cell and Molecular Biology 3 Section of Gene Function and Regulation The Institute of Cancer Research Chester Beatty Laboratories 237 Fulham Road London SW3 6JB United Kingdom 4 Cancer Research UK Centre for Cancer Therapeutics The Institute of Cancer Research 15 Cotswold Road Sutton SM2 5NG United Kingdom 5 Cancer Genome Project The Wellcome Trust Sanger Institute Wellcome Trust Genome Campus Hinxton United Kingdom Summary Over 30 mutations of the B RAF gene associated with human cancers have been identified the majority of which are located within the kinase domain Here we show that of 22 B RAF mutants analyzed 18 have elevated kinase activity and signal to ERK in vivo Surprisingly three mutants have reduced kinase activity towards MEK in vitro but by activating C RAF in vivo signal to ERK in cells The structures of wild type and oncogenic V599EB RAF kinase domains in complex with the RAF inhibitor BAY43 9006 show that the activation segment is held in an inactive conformation by association with the P loop The clustering of most mutations to these two regions suggests that disruption of this interaction converts B RAF into its active conformation The high activity mutants signal to ERK by directly phosphorylating MEK whereas the impaired activity mutants stimulate MEK by activating endogenous C RAF possibly via an allosteric or transphosphorylation mechanism Introduction The RAF MEK ERK signal transduction pathway is a conserved RAS activated protein kinase cascade that regulates cell growth proliferation and differentiation in response to growth factors cytokines and hormones Correspondence david barford icr ac uk D B richard marais icr ac uk R M 6 These authors contributed equally to this work Robinson and Cobb 1997 The transforming activities of the viral oncogenic forms of RAS and RAF were key to their discovery Rapp et al 1983 and RAF was the first effector identified downstream of RAS Avruch et al 2001 Malumbres and Barbacid 2003 RAF activation is initiated by RAS GTP association with the RAS binding domain RBD situated within the N terminal regulatory region of the kinase Concomitant conformational changes and recruitment to the cell membrane promote changes in RAF phosphorylation that combine to stimulate its serine threonine kinase activity triggering sequential phosphorylation and activation of MEK and ERK Kolch 2000 Morrison and Cutler 1997 The three functional RAF proteins in humans A RAF B RAF and C RAF also termed c Raf 1 are dependent on activation segment phosphorylation for activity Chong et al 2001 Zhang and Guan 2000 However the details of their regulatory mechanisms differ because C RAF and A RAF require additional serine and tyrosine phosphorylation within the N region of the kinase domain for full activity Mason et al 1999 and B RAF has a much higher basal kinase activity than either A RAF or C RAF Communication between RAF MEK and ERK requires the scaffolding protein KSR1 a RAF homolog that is devoid of kinase activity Morrison 2001 Constitutive activation of the RAS ERK signaling pathway is common to numerous cancers Approximately 15 of human cancers have activating RAS mutations Malumbres and Barbacid 2003 and recently mutations in B RAF were identified in a large scale screen for genes mutated in human cancer Davies et al 2002 Somatic mutations of B RAF are associated with 60 of malignant melanomas and occur with moderate to high frequency in colorectal Rajagopalan et al 2002 ovarian Singer et al 2003 and papillary thyroid carcinomas Brose et al 2002 Cohen et al 2003 implicating activating oncogenic mutations of B RAF as critical promoters of malignancy Significantly B RAF and RAS mutations are restricted to the same tumor types usually in a mutually exclusive fashion suggesting that these genes are on the same oncogenic signaling pathway and that RAS acts to activate B RAF in these tumors Sequence analysis of the B RAF gene associated with human cancers has identified over 30 single site missense mutations mostly within the kinase domain The mechanism of oncogenic activation of B RAF therefore differs fundamentally from that of v Raf a retroviral oncogene derived from C RAF The constitutive activity and high transforming potential of v Raf most likely results from loss of the autoinhibitory N terminal region combined with targeting to the plasma membrane Most of the mutations of B RAF are clustered to two regions the glycine rich P loop of the N lobe and the activation segment and flanking regions A Glu for Val substitution at residue 599 in the activation segment adjacent to the conserved DFG motif accounts for 90 of B RAF mutations in human cancers The V599E mutant of B RAF possesses the hallmarks of a conventional oncogene The kinase activity of this mutant protein is greatly ele Cell 856 vated it constitutively stimulates ERK activity in vivo independent of RAS and it potently transforms NIH3T3 cells Interestingly the conserved regulatory phosphorylation sites within the activation segment of B RAF Thr598 and Ser601 flank Val599 leading to the suggestion that the Glu substitution at this position functions as a phospho mimetic Davies et al 2002 Analysis of three other oncogenic mutants of B RAF showed that they stimulate kinase activity in a manner similar to V599E B RAF Davies et al 2002 Intriguingly however extensive analysis of B RAF mutations in cancer shows that seven of the mutations involve highly conserved or invariant residues in the catalytic domain Davies et al 2002 Yuen et al 2002 that in other kinases are known to be required for optimal catalytic activity Johnson et al 1996 Manning et al 2002 raising the question of how these mutants promote tumorigenesis To investigate the mechanisms by which mutant oncogenic forms of B RAF promote cancer we have examined a panel of 22 mutants We show that eighteen mutants activate B RAF in vitro and stimulate ERK signaling in vivo conforming to the conventional model of an activating oncogene However four mutants have reduced kinase activity in vitro but