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ARAF Acts As a Scaffold to Stabilize BRAF:CRAF Heterodimers

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ARAF Acts As a Scaffold to Stabilize BRAF:CRAF Heterodimers Oncogene (2013) 32, 3207–3212 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc 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).
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