ARAF Acts As a Scaffold to Stabilize BRAF:CRAF Heterodimers

SHORT COMMUNICATION ARAF acts as a scaffold to stabilize BRAF:CRAF heterodimers

AP Rebocho1,2 and R Marais1,3

The RAF proteins are cytosolic protein kinases that regulate cell responses to extracellular signals. There are three RAF proteins in cells, ARAF, BRAF and CRAF, and recent studies have shown that the formation of complexes by these different isoforms has an important role in their activation, particularly in response to RAF inhibitors. Here, we investigated the role of ARAF in cancer cell signaling and examined the role of ARAF in mediating paradoxical activation of the MAPK pathway in cells treated with RAF inhibitors. We show that two mutations that occur in ARAF in cancer inactivate the kinase. We also show that ARAF is not functionally redundant with CRAF and cannot substitute for CRAF downstream of RAS. We further show that ARAF binds to and is activated by BRAF and that ARAF also forms complexes with CRAF. Critically, ARAF seems to stabilize BRAF:CRAF complexes in cells treated with RAF inhibitors and thereby regulate cell signaling in a subtle manner to ensure signaling efﬁciency.

Oncogene (2013) 32, 3207–3212; doi:10.1038/onc.2012.330; published online 27 August 2012 Keywords: ARAF; BRAF; CRAF; protein complexes; paradoxical activation

INTRODUCTION dimerization, leading to the formation of BRAF:CRAF heterodimers The RAF proteins are serine-threonine-specific protein kinases that and CRAF:CRAF homodimers containing a drug-bound and a activate the MEK/ERK cascade downstream of RAS. In normal cells, drug-free partner. Critically, the drug-bound partner activates this pathway is activated by extracellular signals and controls the drug-free partner through scaffold functions or by inducing proliferation, survival, senescence and differentiation, whereas in a conformational change. cancer, it is constitutively activated, so proliferation and survival These studies reveal the importance of RAF interactions in MEK/ are the favored outcomes. There are three RAF genes in humans, ERK pathway activation, but, although the importance of namely, ARAF, BRAF and CRAF, and although BRAF is mutated in BRAF:CRAF heterodimers and CRAF:CRAF homodimers have been B7% of cancers, ARAF and CRAF mutations are rare.1,2 This is due explored in some depth, the role of ARAF in these processes is less to a fundamental difference in RAF isoform regulation mediated well understood. We therefore studied the role of ARAF in cancer by a motif called the N-region. ARAF and CRAF require cell signaling and ARAF function in paradoxical activation of the phosphorylation of conserved serines and tyrosines in the MAPK pathway in cells treated with RAF inhibitors. N-region for activation, whereas in BRAF, the serine is constitutively phosphorylated and the tyrosine is substituted with an aspartic acid. Thus BRAF, unlike ARAF or CRAF, is primed RESULTS AND DISCUSSION for activation and can be converted into an active oncogene by To explore ARAF signaling in tumorigenesis, we first characterized single-point mutations.3 two ARAF mutants identified in human cancer. G331CARAF was The most common mutation in BRAF in cancer is substitution of identified in a colon carcinoma and A451TARAF in Molt–4 valine 600 (V600) for glutamic acid.2,4 V600EBRAF has elevated lymphoblastic leukemia cells.17,18 We previously demonstrated kinase activity and is a confirmed driver oncogene in cancer. that ARAF could be activated by G12VHRAS and Y527FSRC,19 but show Importantly, BRAF inhibitors, such as vemurafenib, mediate here that G331CARAF and A451TARAF were not activated by these impressive clinical responses in melanoma patients whose oncogenes (Figure 1a). Next, we tested whether ARAF, like CRAF,9 tumors express V600EBRAF.5–7 Over 100 mutations have been could be activated by G596RBRAF. G596RBRAF stimulated robust described in BRAF in cancer, the majority of which possess activation of ARAF but did not activate G331CARAF or A451TARAF elevated kinase activity. However, some, such as G596RBRAF, (Figure 1b). We confirmed that the activity measured in these have impaired kinase activity and cannot phosphorylate MEK experiments was of ARAF, because we did not detect RAF directly.8 Nevertheless they still activate the pathway because they activity when using kinase-dead forms of ARAF (Supplementary bind to and activate CRAF in a RAS-independent manner.9,10 Figure S1a). Furthermore, it is CRAF and not BRAF that transmits signals from The inactivation of ARAF by G331C and A451T was unexpected. oncogenic RAS to MEK.11–13 Thus, although CRAF mutations are G331 is between the glycine-rich loop and catalytic lysine of the rare in human cancer, CRAF does signal in some contexts and is a kinase domain and A451 is adjacent to T452, a conserved validated therapeutic target. activation segment phosphorylation site.20,21 These residues are Recent studies have identified a paradox with RAF inhibitors, conserved in BRAF and CRAF and the crystal structure of BRAF because although they inhibit the MEK/ERK pathway in cells did not reveal why these substitutions would be inactivating expressing V600EBRAF, they activate the pathway in cells expres- (Supplementary Figure S1b), so we engineered the equivalent sing oncogenic RAS.14–16 This is because RAF inhibitors drive RAF mutations into BRAF (G478CBRAF, A598TBRAF). Notably, G478CBRAF

1Signal Transduction Team, Division of Cancer Biology, The Institute of Cancer Research, London, UK. Correspondence: Professor R Marais, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. E-mail: [email protected] 2Current address: Department of Biochemistry, Cambridge University, 80 Tennis Court Road, Cambridge CB2 1GA, UK. 3Current address: Paterson Institute for Cancer Research, University of Manchester, Manchester, UK. Received 2 March 2012; revised 7 June 2012; accepted 10 June 2012; published online 27 August 2012 ARAF is a scaffold protein AP Rebocho and R Marais 3208

Figure 1. ARAF cancer mutants are inactive. (a) ARAF kinase activity from COS7 cells expressing empty vector control (EV), ARAF, G331CARAF or A451TARAF with G12VHRAS (RAS) and Y527FSRC (SRC) was as described.9,19 The relative concentration of exogenous RAF proteins was determined by quantitative western blot to ensure that equivalent amounts of proteins were immunoprecipitated in the in vitro kinase assays. Data are from a single assay in triplicate with error bars to represent standard error (s.e.) from the mean and a minimum of two independent replicates. (b) ARAF kinase activity from COS7 cells expressing empty vector control (EV), ARAF, G331CARAF and A451TARAF with G596RBRAF. (c) BRAF kinase activity from COS7 cells expressing BRAF, G478CBRAF or A598TBRAF. (d) ARAF kinase activity from COS7 cells expressing empty vector control (EV), ARAF with G478CBRAF, A598TBRAF or G596RBRAF. (e) Colony formation in NIH3T3 cells expressing ARAF (WT), G331CARAF (G331C), A451TARAF (A451T) or G12VHRAS (RAS) was performed as previously described.3 Error bars represent s.e. from the means. (f) Whole cell lysates (30 mg) were prepared in NP40 buffer as described9 and western blotted for CRAF (610152; BD Transduction Laboratories, San Jose, CA, USA), ARAF (C–20; Santa Cruz Biotechnology, Heidelberg, Germany), phospho-ERK (ppERK; M8159; Sigma-Aldrich, St Louis, MO, USA) and total ERK2 (C–14; Santa Cruz) in D04 cells treated with scrambled control (Scr) or CRAF siRNA and co-transfected with empty vector control G331C E439K A451T (EV), ARAF, ARAF (G331C), ARAF (E439K) or ARAF (A451T). Simultaneous transfection of siRNA (5 nM) and DNA (400 ng) was performed using effectene according to manufacturer’s instructions (Quiagen, Hilden, Germany) and using the following siRNA sequences: CRAF: 50-GCACGCUUAGAUUGGAAUA-30; scrambled control: 50-AAGUCCAUGGUGACAGGAGAC-30.(g) ARAF kinase activity from COS7 cells expressing empty vector control (EV), ARAF and E439KARAF (E439K) with G12VHRAS (RAS) and Y527FSRC (SRC) or G596RBRAF.

Oncogene (2013) 3207 – 3212 & 2013 Macmillan Publishers Limited ARAF is a scaffold protein AP Rebocho and R Marais 3209 and A598TBRAF were also inactive (Figure 1c), but unlike G596RBRAF, A451TARAF and G331CARAF do not bind to BRAF (data not shown), they did not activate ARAF (Figure 1d). providing a possible explanation for their lack of activity. Next, we tested G331CARAF and A451TARAF in cells. Unlike ARAF mutations are rare in cancer and our data reveals that G12VHRAS, G331CARAF and A451TARAF did not transform NIH3T3 they show an unexpected lack of activity, suggesting they are cells (Figure 1e). We therefore expressed them in D04 cells, a either passenger mutations or that their oncogenic activity is only melanoma line expressing Q61LNRAS. ARAF, G331CARAF and revealed in specific signaling contexts. We also show that ARAF A451TARAF overexpression did not increase ERK activity in D04 cannot substitute for CRAF, suggesting that they perform distinct cells (Figure 1f). Previously,11 we demonstrated that CRAF functions in RAS mutant cells. We previously reported that BRAF depletion by RNA interference suppressed ERK activity in RAS binds to and activates CRAF,9 so tested whether BRAF also bound mutant cells and show here that ARAF, G331CARAF and A451TARAF to ARAF. Myc-tagged ARAF and HA-tagged BRAF were expressed overexpression did not rescue ERK activity when CRAF was in COS7 cells and ARAF was immunoprecipitated and probed for depleted (Figure 1f). As a control, we also tested whether an BRAF by western blot. We show that BRAF bound to ARAF weakly, activated version of ARAF could rescue ERK activity in these cells. but that this binding was substantially increased by G12VHRAS For this, we generated E439KARAF, the ARAF equivalent of (Figure 2a). Notably, V600EBRAF and G596RBRAF bound to ARAF E478KCRAF and E586KBRAF, both of which show elevated kinase robustly, even in the absence of oncogenic RAS (Figure 2a). activity.2,3,9 We verified that E439KARAF was strongly activated by We confirmed these findings by immunoprecipitating HA-BRAF G12VHRAS and Y527FSRC and by G596RBRAF (Figure 1g). Never- and showing that myc-ARAF was bound to it (Figure 2a). theless, E439KARAF only partially rescued ERK activity in CRAF- Next, we tested whether endogenous ARAF bound to depleted D04 cells (Figure 1f). endogenous BRAF. We observed a protein of B90kDa that bound The data above show that G331CARAF and A451TARAF lacked to ARAF in WM266.4 melanoma (V600DBRAF) and HCT116 color- enzymatic and biological activity. A451 is in the activation segment, ectal cancer (G13DKRAS) cells, but was lost when the cells were adjacent to the phosphorylation site at T45221 and notably, the treated with BRAF targeting small-interfering RNA (siRNA) probes corresponding region in BRAF is a hotspot for mutations that (Figure 2b). Similarly, we observed a protein of B70kDa that activate BRAF.22 It was therefore surprising that the A451T bound to BRAF, but was lost when ARAF was depleted by siRNA substitution was not activating, but others have suggested that (Figure 2c). These data confirmed that endogenous BRAF and introduction of a hydroxyl group into this region in BRAF would ARAF bound to each other in BRAF and RAS mutant cells. We stabilize the inactive conformation of the protein by altering local previously demonstrated that RAS recruited BRAF and CRAF to the electrostatic interactions.23 Accordingly, we confirmed here that plasma membrane,19 so tested whether RAS also recruits ARAF to A598TBRAF lacked kinase activity. G331 is also in the kinase domain the membrane. We confirmed that ARAF was cytosolic in the and again the BRAF equivalent (G478CBRAF) was inactive. It is unclear absence of oncogenic RAS, but membrane bound in its why this mutant lacked activity, but our preliminary data show that presence (Figure 2d). In contrast, ARAF remained cytosolic in the

Figure 2. ARAF is a BRAF effector. (a) Western blot for myc-ARAF (9B11; Cell Signaling, Boston, MA, USA) or HA-BRAF (H3663; Sigma-Aldrich) in myc or HA immunoprecipitates (Myc IP; HA IP) or cell lysates from COS cells expressing empty vector (EV), myc-ARAF (ARAF) or HA-tagged versions of BRAF, G596RBRAF, V600EBRAF or G12VHRAS (RAS). Proteins were immunoprecipitated as described,14 using 12CA5 (ICR Hybridoma Unit) for HA-tagged proteins or anti-myc (ab9106; Abcam, Cambridge, United Kingdom) for myc-transfected proteins. (b, c) Western blots for endogenous ARAF, BRAF (F7; Santa Cruz) and ERK2 (loading control) in ARAF (b; ARAF IP) or BRAF (c; BRAF IP) immunoprecipitates or cell lysates from WM266.4 or HCT116 cells transfected with BRAF (b; BRAF1: 50-AGAAUUGGAUCUGGAUCAU-30; BRAF2: 50- UCAGUAAGGUACGGAGUAA-30), ARAF (c; ARAF1: 50-ACCGAGAUCUCAAGUCUAAUU-30; ARAF2: 50-CUACGACUCUCUAGACAAGUU-30) siRNAs or scrambled control (Scr). RNA interference-mediated depletion was performed using lipofectamine (Invitrogen, Paisley, Scotland) as described.19 Endogenous proteins were immunoprecipitated using anti-ARAF (4432; Cell Signaling) or anti-BRAF (C–19; Santa Cruz) antibodies. (d) Western blots for myc-ARAF, HA-BRAF, HRAS (F235; Santa Cruz), tubulin (cytosolic control; T5168; Sigma-Aldrich) and caveolin1 (membrane control; N–20; Santa Cruz) in membrane fraction (MEM) or cytosolic fraction (CYT) of COS cells expressing myc-ARAF (ARAF), HA-BRAFG596R (G596RBRAF) and G12VHRAS (RAS). The spaces indicate discontinuous sections of the same gel. Cell fractionation assays were as described.10

& 2013 Macmillan Publishers Limited Oncogene (2013) 3207 – 3212 ARAF is a scaffold protein AP Rebocho and R Marais 3210 presence of G596RBRAF (Figure 2d). Thus, ARAF bound to WTBRAF at binding to BRAF and/or CRAF. We treated D04 cells with the BRAF the plasma membrane in a RAS-dependent manner, but bound to selective inhibitor SB590885 and the pan-RAF inhibitor sorafenib, and was activated by cytosolic G596RBRAF in a RAS-independent immunoprecipitated endogenous ARAF and probed for BRAF. We manner. We conclude that like CRAF, ARAF is a BRAF effector observed that ARAF formed a constitutive complex with BRAF in protein. D04 cells (Figure 3a), but that this interaction was further Recent studies have shown that RAF inhibitors drive CRAF enhanced by RAF inhibitors in a dose-dependent manner, binding to BRAF,14,15 so we tested whether they also drove ARAF conﬁrming previous ﬁndings.15 Note that ARAF binding to BRAF

Figure 3. RAF inhibitors activate ARAF and enhance its binding to other RAF isoforms. (a) Western blots for endogenous ARAF, BRAF, CRAF, phospho-MEK (ppMEK; 9121; Cell Signaling), phospho-ERK (ppERK) or ERK2 (loading control) in ARAF immunoprecipitates (ARAF IP) or cell lysates from D04 cells treated for 2 h with DMSO ( À ), SB590885 (SB; Symansis, Timaru, New Zealand) or Sorafenib (SF) at the indicated concentrations. All drugs were prepared in DMSO. The dotted lines indicate discontinuous sections of the same gel. (b) Western blots for myc- tagged ARAF and HA-tagged BRAF in myc immunoprecipitates (Myc IP) or cell lysates from COS7 cells expressing empty vector control (EV), myc-ARAF (ARAF), myc-ARAFR52L (R52L), HA-BRAF (BRAF), HA-BRAFR188L (R188L) or G12VHRAS (RAS). (c) ARAF kinase activity from COS7 cells expressing empty vector control (EV), ARAF, R52LARAF (R52L), BRAF, R188LBRAF (R188L) or G12VHRAS (RAS). (d) Western blots for endogenous ARAF and CRAF in ARAF immunoprecipitates (ARAF IP) or cells lysates from D04 and MM415 cells treated with DMSO ( À ), Sorafenib (SF; 10 mM), SB590885 (SB; 0.1 mM) or PD184352 (PD; 1 mM)for2h.(e) Endogenous ARAF kinase activity in D04 and WM266.4 cells treated with SB590885 (0.1 mM) for various time points. (f) ARAF kinase activity in Araf null mouse embryonic fibroblasts ( À / À ArafMEF) expressing empty vector control (EV), K336M D447A ARAF, ARAF (K336M) or ARAF (D447A) treated with SB590885 (0.1 mM) for 2 h. For transient expression studies, MEFs were transfected using the Amaxa Nucleofactor System according to manufacturer’s instructions (Lonza, Basel, Switzerland). (g) Endogenous ARAF kinase activity from Braf null mouse embryonic fibroblasts ( À / À BrafMEF) expressing empty vector control (EV), BRAF or T529NBRAF and treated with DMSO ( À ) or SB590885 (SB; 0.1 mM) for 2 h. ARAF activation was significantly different for all the conditions tested (P ¼ 0.0013).

Oncogene (2013) 3207 – 3212 & 2013 Macmillan Publishers Limited ARAF is a scaffold protein AP Rebocho and R Marais 3211 was constitutive in BRAF mutant A375P and WM266.4 melanoma confirmed that the MEK inhibitor PD184352 stabilized ARAF:CRAF cells and was not enhanced by sorafenib, SB590885 or PD184352 complexes in SB590885–treated cells (Figure 3d). (Supplementary Figure S2a), confirming that these drugs did not Next, we examined whether ARAF was activated by drug- increase ARAF binding to mutant BRAF. induced RAF dimerization. SB590885 activated ARAF robustly in We previously demonstrated that BRAF:CRAF dimerization D04 cells (Figure 3e), albeit less efficiently than it activated CRAF required both BRAF and CRAF binding to RAS,14 so tested (Supplementary Figure S2b). To confirm the activation of ARAF by whether ARAF binding to RAS was also required. We generated SB590885, we expressed kinase-dead forms of ARAF in Araf null an ARAF mutant (R52LARAF) that cannot bind to RAS and mouse embryonic fibroblasts (MEFs) and showed that these mutants found that this mutant still bound to BRAF in the presence of were not activated (Figure 3f, Supplementary Figure S2c). Critically, oncogenic RAS (Figure 3b). Notably however, the corresponding ARAF activation was impaired in Braf null MEFs (Supplementary RAS-binding-deficient BRAF mutant (R188LBRAF) did not bind to Figure S2d), but recovered when BRAF was re-expressed in these ARAF, showing that the BRAF:ARAF interaction required only BRAF cells (Figure 3g; Supplementary Figure S2e). Note, when T529NBRAF, to bind to RAS (Figure 3b). These results were corroborated by a BRAF mutant that does not bind to SB590885, was expressed in kinase assays where we show that ARAF activation by BRAF Braf null cells, ARAF was not activated (Figure 3g). This demonstrates required BRAF binding to RAS, but not ARAF binding to RAS that it is binding of the drugs to BRAF rather than ARAF that drives (Figure 3c). ARAF activation. Finally, we did not observe ARAF activation by While performing these studies, we observed that ARAF also SB590885 in BRAF mutant WM266.4 cells (Figure 3e). bound to CRAF (Figure 3a). Sorafenib and, albeit less efficiently, Our data confirmed that RAF inhibitors activated ARAF but also SB590885 induced ARAF binding to CRAF. However, in contrast to demonstrated that ARAF and CRAF bound to BRAF with different ARAF binding to BRAF, ARAF binding to CRAF in untreated cells was characteristics. Specifically, whereas CRAF binding to BRAF required variable and, in general, ARAF did not bind to CRAF in untreated cells CRAF binding to RAS,14 ARAF did not need to bind to RAS to bind to (Figure 3d), although weak binding was occasionally seen (Figure 3a). BRAF or be activated. Moreover, although RAF inhibitors increased We concluded that ARAF binding to CRAF was less robust that ARAF ARAF and CRAF binding to BRAF, ARAF bound to BRAF weakly even bindingtoBRAF,andinagreementwithpreviousdatashowingthat in untreated cells whereas CRAF did not. Thus, ARAF and CRAF seem ERK destabilized RAF dimers by feedback phosphorylation,24 we to be paradoxically activated by distinct mechanisms.

Figure 4. ARAF stabilizes BRAF interaction with CRAF. (a) Western blot for ARAF, BRAF, CRAF, phospho-MEK (ppMEK), phospho-ERK (ppERK) and ERK2 (loading control) in D04 cells transfected with non-speciﬁc control (Scr), or ARAF (A), or BRAF (B), or CRAF (C) siRNA as described.28 After 72 h, cells were treated with DMSO ( À ) or SB590885 ( þ ; 0.1 mM) for 2 h. (b) Western blots for endogenous ARAF, BRAF, CRAF, phospho- MEK (ppMEK), phospho-ERK (ppERK) and total ERK2 (loading control) in CRAF immunoprecipitates (CRAF IP; C–20; Santa Cruz) and cell lysates from D04 cells transfected with scrambled control (Scr) or two ARAF (ARAF1, ARAF2) siRNA probes. Seventy-two hours after transfection, cells were treated with DMSO ( À ) or SB590885 ( þ ; 0.1 mM) for 2 h. (c) Western blots for myc-tagged CRAF, HA-tagged CRAF and total ERK2 (loading control) in myc-CRAF immunoprecipitates (Myc IP) or cell lysates from D04 cells expressing empty vector control (EV), myc-CRAF or HA-CRAF and treated with DMSO ( À ) or SB590885 ( þ ; 0.1 mM) for 2 h. For transient expression studies, D04 cells were transfected using the Amaxa Nucleofactor System. (d) Western blots for myc-tagged BRAF, HA-tagged BRAF and ERK2 (loading control) in myc-BRAF immunoprecipitates (Myc IP) or total cell lysates from D04 cells expressing empty vector control (EV), myc-BRAF or HA-BRAF and treated with DMSO ( À )or SB590885 ( þ ; 0.1 mM) for 2 h. (e) Western blots for myc-tagged ARAF, HA-tagged ARAF and total ERK2 (loading control) in myc-ARAF immunoprecipitates (Myc IP) or cell lysates from D04 cells expressing empty vector control (EV), myc-ARAF or HA-ARAF and treated with DMSO ( À ) or SB590885 ( þ ; 0.1 mM) for 2 h.

& 2013 Macmillan Publishers Limited Oncogene (2013) 3207 – 3212 ARAF is a scaffold protein AP Rebocho and R Marais 3212 We next examined the role of ARAF in paradoxical activation of 2 Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S et al. Mutations of the the MEK/ERK pathway. Depletion of ARAF in D04 cells did not impair BRAF gene in human cancer. Nature 2002; 417: 949–954. MEK/ERK activity (Figure 4a). Similar responses were observed in 3 Emuss V, Garnett M, Mason C, Marais R.. Mutations of C-RAF are rare in human three other NRAS mutant cell lines (Supplementary Figure S3a), and cancer because C-RAF has a low basal kinase activity compared with B-RAF. ARAF depletion did not suppress MEK/ERK signaling in BRAF mutant Cancer Res 2005; 65: 9719–9726. cells (Supplementary Figure S3b). As already shown (Figure 1f), CRAF 4 Wellbrock C, Karasarides M, Marais R. The RAF proteins take centre stage. Nat Rev depletion suppressed MEK/ERK activity in D04 cells (Figure 4a). Mol Cell Biol 2004; 5: 875–885. Critically, and despite not affecting ERK activity alone, ARAF 5 Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 2010; 363: depletion cooperated with CRAF depletion to further inhibit MEK/ 809–819. ERK activity (Figure 4a). These data confirmed that ARAF and CRAF 6 Bollag G, Hirth P, Tsai J, Zhang J, Ibrahim PN, Cho H et al. Clinical efficacy of a RAF have distinct roles in MEK/ERK signaling in RAS mutant cells. inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature 2010; Importantly, when ARAF was depleted in DO4 cells, we observed a 467: 596–599. strong reduction in CRAF binding to BRAF (Figure 4b), suggesting 7 Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J et al. that ARAF stabilized BRAF binding to CRAF. Surprisingly, the Improved survival with vemurafenib in melanoma with BRAF V600E mutation. destabilization of the BRAF:CRAF complex in ARAF-depleted cells N Engl J Med 2011; 364: 2507–2516. did not reduce paradoxical activation of MEK and ERK in SB590885- 8 Dhomen N, Marais R. New insight into BRAF mutations in cancer. Curr Opin Genet treated cells (Figure 4b). However, paradoxical activation of MEK and Dev 2007; 17: 31–39. 16 9 Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM et al. Mechanism ERK can be mediated by CRAF:CRAF homodimers, so we of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. transfected myc and HA-tagged versions of CRAF into D04 cells, Cell 2004; 116: 855–867. immunoprecipitated the myc-tag proteins and probed for the 10 Garnett MJ, Rana S, Paterson H, Barford D, Marais R. Wild-type and mutant B-RAF presence of the HA-tagged protein. Critically, we show that activate C-RAF through distinct mechanisms involving heterodimerization. Mol SB590885 promoted the formation of CRAF homodimers Cell 2005; 20: 963–969. (Figure 4c) and, for the first time, that SB590885 also induced BRAF 11 Dumaz N, Hayward R, Martin J, Ogilvie L, Hedley D, Curtin JA et al. In melanoma, (Figure 4d) and ARAF (Figure 4e) homodimerization. RAS mutations are accompanied by switching signaling from BRAF to CRAF and Although signaling can be rewired amongst RAF isoforms in disrupted cyclic AMP signaling. Cancer Res 2006; 66: 9483–9491. drug-resistant BRAF mutant cells,25 we show here that ARAF and 12 Blasco RB, Francoz S, Santamaria D, Canamero M, Dubus P, Charron J et al. c-Raf, but not B-Raf, is essential for development of K-Ras oncogene-driven non-small CRAF cannot compensate for each other in a RAS mutant back- cell lung carcinoma. Cancer Cell 2011; 19: 652–663. ground, and therefore perform distinct functions. We also show that 13 Karreth FA, Frese KK, DeNicola GM, Baccarini M, Tuveson DA. C-Raf Is Required for ARAF is a BRAF effector protein that was activated by wild-type BRAF the Initiation of Lung Cancer by K-RasG12D. Cancer Discovery 2011; 1: 128–136. in a RAS-dependent manner, but was activated by mutant BRAF in a 14 Heidorn SJ, Milagre C, Whittaker S, Nourry A, Niculescu-Duvas I, Dhomen N et al. RAS-independent manner. Notably, however, we demonstrated that Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression unlike CRAF, ARAF did not need to bind to RAS to bind to wild-type through CRAF. Cell 2010; 140: 209–221. BRAF. We further demonstrated that in some cells ARAF stabilized 15 Hatzivassiliou G, Song K, Yen I, Brandhuber BJ, Anderson DJ, Alvarado R et al. RAF CRAF binding to BRAF, but our preliminary data suggests that ARAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance was not essential for CRAF binding to BRAF in all cells, possibly growth. Nature 2010; 464: 431–435. 16 Poulikakos PI, Zhang C, Bollag G, Shokat KM, Rosen N. RAF inhibitors transactivate because other proteins, including the kinase suppressor of RAS (KSR) RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 2010; 464: can also perform this function and so mask the contribution from 427–430. ARAF. Indeed, the scaffold function of proteins such as ARAF and 17 Lee JW, Soung YH, Kim SY, Park WS, Nam SW, Min WS et al. Mutational analysis KSR may be highly complex; in some cells KSR was reported to of the ARAF gene in human cancers. Apmis 2005; 113: 54–57. enhance CRAF activation by RAF inhibitors,26 whereas in other cells 18 Greenman C, Stephens P, Smith R, Dalgliesh GL, Hunter C, Bignell G et al. Patterns KSR was reported to antagonize CRAF activation.27 of somatic mutation in human cancer genomes. Nature 2007; 446: 153–158. Recent studies have established an unexpected complexity in 19 Marais R, Light Y, Paterson HF, Mason CS, Marshall CJ. Differential regulation of RAF signaling that makes it difficult to assign specific functions to Raf–1, A-Raf, and B-Raf by oncogenic ras and tyrosine kinases. J Biol Chem 1997; individual RAF isoforms and, accordingly, we show that the 272: 4378–4383. 20 Zhang BH, Guan KL. Activation of B-Raf kinase requires phosphorylation of the contribution of ARAF to RAS signaling was only apparent when conserved residues Thr598 and Ser601. EMBO J 2000; 19: 5429–5439. CRAF was depleted. Nevertheless, we confirm that ARAF is a BRAF 21 Chong H, Lee J, Guan KL. Positive and negative regulation of Raf kinase activity effector protein that is paradoxically activated by RAF inhibitors and function by phosphorylation. Embo J 2001; 20: 3716–3727. and we reveal an additional level of complexity by showing that, 22 Garnett MJ, Marais R. Guilty as charged: B-RAF is a human oncogene. Cancer Cell in some cells, ARAF stabilizes BRAF:CRAF complexes to sustain 2004; 6: 313–319. signaling efficiency. 23 Moretti S, De Falco V, Tamburrino A, Barbi F, Tavano M, Avenia N et al. Insights into the molecular function of the inactivating mutations of B-Raf involving the DFG motif. Biochim Biophys Acta 2009; 1793: 1634–1645. 24 Ritt DA, Monson DM, Specht SI, Morrison DK. Impact of feedback phosphorylation CONFLICT OF INTEREST and Raf heterodimerization on normal and mutant B-Raf signaling. Mol Cell Biol The authors declare no conflict of interest. 2010; 30: 806–819. 25 Villanueva J, Vultur A, Lee JT, Somasundaram R, Fukunaga-Kalabis M, Cipolla AK et al. Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in ACKNOWLEDGEMENTS melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell 2010; 18: 683–695. This work was funded by Fundaçaõ para a Cieˆncia e Tecnologia (SFRH/BD/15904/ 26 Hu J, Yu H, Kornev AP, Zhao J, Filbert EL, Taylor SS et al. Mutation that blocks ATP 2005), the Institute of Cancer Research and Cancer Research UK (Ref: C107/A10433). binding creates a pseudokinase stabilizing the scaffolding function of kinase We thank Professor Caroline Springer for providing sorafenib and PD184352. suppressor of Ras, CRAF and BRAF. Proc Natl Acad Sci USA 2011; 108: 6067–6072. 27 McKay MM, Ritt DA, Morrison DK. RAF inhibitor-induced KSR1/B-RAF binding and its effects on ERK cascade signaling. Curr Biol 2011; 21: 563–568. REFERENCES 28 Packer LM, Rana S, Hayward R, O’Hare T, Eide CA, Rebocho A et al. Nilotinib and 1 Mercer KE, Pritchard CA. Raf proteins and cancer: B-Raf is identified as a MEK Inhibitors Induce Synthetic Lethality through Paradoxical Activation of RAF in mutational target. Biochim Biophys Acta 2003; 1653: 25–40. 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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2013) 3207 – 3212 & 2013 Macmillan Publishers Limited