Mardiyyah/TAPT_data_V2_split · Datasets at Hugging Face

PMCID stringclasses 24 values	Title stringclasses 24 values	Sentences stringlengths 2 40.7k
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	They were highly proliferative as evidenced by large numbers of mitotic figures in the superficial and deep aspects of the lesions (∼6 mitosis/10HPF; Figure 5H) and positive staining for Ki67 throughout (Figure 5I).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The tumors were strongly and diffusely positive for S100 (Figure 6A) and expressed the melanocyte markers tyrosinase, Dct, Pax3, and silver (Figure 6B), consistent with a diagnosis of melanoma.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Genomic DNA analysis of the tumors and cell lines derived from them confirmed that Braf had been recombined to Braf (Figure 6C).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	However, for technical reasons we could not detect Kras recombination (data not shown), so used RT-PCR to amplify and sequence Kras mRNA.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	We show that only wild-type Kras is expressed in the kidneys, whereas the tumors expressed both wild-type Kras and Kras (Figure 6D).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Importantly, we show constitutive binding of Braf to Craf in cells from the Kras/Braf tumors (Figure 6E).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	As a control, we used cells from melanoma induced by Kras overexpression.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Briefly, when Kras was overexpressed in melanocytes in mice using the β-actin promoter (β-actin:LSL:Kras; Meuwissen et al., 2001), it induced rapid onset melanoma (median time to onset 2 months, 100% penetrance within 3 months) in the absence of Braf (manuscript submitted).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Importantly, in cells from these tumors, Braf does not bind to Craf (Figure 6E).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Thus, it is only kinase-dead Braf and not wild-type Braf that binds to Craf in the presence of oncogenic Kras.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	In this study, we show that inhibition of BRAF by chemical or genetic means in the presence of oncogenic or growth-factor activated RAS induces BRAF binding to CRAF, leading to CRAF hyperactivation and consequently elevated MEK and ERK signaling.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The mechanism we describe is another paradigm of RAF activation downstream of RAS and based on our findings, we propose the following mechanism by which this occurs.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	We posit that in RAS mutant cells, BRAF maintains itself in an inactive conformation through its own kinase activity, either through auto-phosphorylation, or by phosphorylating a partner protein that then keeps it inactive (Figure 7A).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	We are currently using mass-spectrometry and mutagenic approaches to elucidate the underlying mechanism.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	We propose that when BRAF is inhibited, it escapes this auto-inhibited state and is recruited to the plasma membrane by RAS, where it forms a stable complex with CRAF.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Critically, we posit that because it is inhibited, BRAF does not directly phosphorylate MEK, but rather it acts as a scaffold whose function is to enhance CRAF activation, thereby allowing CRAF to hyperactivate the pathway (Figure 7B).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	We do not know the stoichiometry of the components in these complexes, but since BRAF and CRAF must both bind to RAS for complex formation, it seems likely that at least two RAS proteins are needed to stimulate formation of the complex (Figure 7B).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	It is unclear why PLX4720 only induces weak binding of BRAF to CRAF, but this may stem from its unique property of displacing the α-C helix of BRAF when it binds (Tsai et al., 2008) and suggests that this helix is important for BRAF binding to CRAF, something that will only be resolved when the BRAF:CRAF crystal structure is solved.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	We have attempted to identify other proteins that may be required to stabilize the BRAF–CRAF complexes.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Our unpublished mutagenesis data suggests that 14-3-3 is required to stabilize these drug-induced complexes (data not shown) and this is consistent with previous observations demonstrating that 14-3-3 mediates BRAF binding to CRAF (Garnett et al., 2005; Rushworth et al., 2006).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Although this appears to contradict our observation that dephosphorylation does not disrupt the complex, because 14-3-3 binds to BRAF and CRAF in a phosphorylation-dependent manner, we presume that 14-3-3 protects these sites from dephosphorylation.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	We have also used RNAi to examine the potential role of other proteins implicated in BRAF-CRAF complex formation or pathway activation, including the scaffold proteins KSR, Sprouty2 and RKTG and the small G protein RHEB, but our preliminary results have not revealed obvious roles for these proteins.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Our studies have parallels to the recently described heterodimers between DRAF and KSR in Drosophila (Rajakulendran et al., 2009).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Notably, flies have only one RAF isoform and it appears to be an ortholog of BRAF rather than ARAF or CRAF.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Our inability to demonstrate an obvious role for KSR in mediating BRAF binding to CRAF or CRAF activation by BRAF suggests that the mechanism underlying dimerization here may be different from those described in flies, but clearly additional studies are required to investigate further the role of scaffold proteins in mediating the phenomena we report.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	In contrast to the BRAF-selective inhibitors, the pan-RAF inhibitors appear to induce paradoxical activation of CRAF.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	They induce BRAF binding to CRAF and CRAF activation, but do not activate MEK–ERK signaling.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	We posit that this is because these agents target both BRAF and CRAF.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Thus, although their inhibition of BRAF will stimulate CRAF activation, they will simultaneously inhibit CRAF (Figure 7C).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	This model is supported by our observation that CRAF converts sorafenib from a pathway inhibitor to a pathway activator and we argue that the paradoxical activation of CRAF by these inhibitors is mediated by BRAF, rather than disrupted feedback inhibition as previously suggested (Hall-Jackson et al., 1999).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Recently, paradoxical activation of PKB/AKT and PKCɛ was also described (Cameron et al., 2009; Okuzumi et al., 2009).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	While ATP-competitive inhibition can block kinase function, they do not block the upstream events that activate the target kinase.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For instance, PKB/AKT inhibitors block the function of this kinase, but occupation of the ATP-pocket by these inhibitors was sufficient to induce the priming phosphorylation usually required for its full activation (Okuzumi et al., 2009).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Inhibitor binding to PKCɛ has been shown to have a similar effect (Cameron et al., 2009).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Importantly, the paradoxical activation of PKB/AKT and PKCɛ did not result in pathway activation because of the continued presence of the inhibitors (Frye and Johnson, 2009).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	In contrast, although BRAF inhibitors also block BRAF kinase activity, this relieves auto-inhibition and results in BRAF hyperphosphorylation, BRAF binding to CRAF, pathway activation and oncogenesis, all presumably because BRAF can heterodimerize with CRAF.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Our study also highlights the critical difference between BRAF-selective and pan-RAF drugs.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Whereas BRAF-selective drugs cause pathway activation in a RAS-dependent manner, this does not occur with pan-RAF drugs.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Our results provide important insight into the genetics of human cancer.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Excluding V600 mutants, D594 mutants are the third most common in BRAF in cancer (34 out of 443 cases or ∼7.7%; www.sanger.ac.uk/genetics/CGP/cosmic/).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Furthermore, as mentioned in the Introduction, while BRAF and RAS mutations are generally mutually exclusive, 4 of the 34 (11.8%) tumors with D594 mutations also have mutations in RAS.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	This is a highly significant enrichment for the coincidence of these mutations (p < 10; Fisher's Exact Test) and suggests a functional interaction.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	We now provide strong circumstantial evidence of such an interaction using transgenic mice.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	By themselves, Braf and Kras do not induce melanoma, but they cooperate to induce rapid onset melanoma.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	This highly significant result (p < 0.0002) provides a rational explanation for the coincidence of these mutations in human cancer.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Furthermore, we show that the BRAF inhibitors also hyperactivate this pathway in growth factor stimulated cells, providing an explanation of why kinase dead BRAF mutations are not always coincident with RAS mutations; presumably in some tumors the cooperating mutation is upstream of RAS.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Our results also suggest several potential mechanisms by which resistance to RAF targeting drugs could develop in patients.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	BRAF mutant tumors could become resistant to BRAF-selective drugs, if they acquire a mutation in RAS or an upstream component that activates RAS, or if the drugs select a population of cells harboring pre-existing mutations in RAS.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Theoretically this would cause BRAF-mediated CRAF activation, which may not only induce resistance, but could potentially promote tumor growth.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	In line with this, increased expression of CRAF can mediate acquired resistance to pan-RAF drugs in BRAF mutant cancer cells in vitro (Montagut et al., 2008), establishing that CRAF can mediate resistance under some circumstances.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Our in vitro studies also suggest that a potential mechanism of resistance in patients with RAS mutant tumors being treated with pan-RAF drugs is acquisition (or selection for cells with pre-existing mutations) of a CRAF mutation such as a gatekeeper mutant that prevents drug binding.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Again this would potentially result in BRAF-mediated activation of CRAF (Figure 7D) and possibly accelerated tumor growth.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Although our studies are restricted to cell lines and transgenic mice, they do have important immediate clinical implications.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	They strongly argue that BRAF-selective inhibitors should not be administered to patients with RAS mutant tumors, because long-term use could accelerate tumor growth.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Intriguingly, 10%–15% of patients treated with BRAF-selective drugs develop squamous cell carcinoma (SCC)(Flaherty et al., 2009; Schwartz et al., 2009).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Although MEK–ERK signaling has not yet been implicated in this response, 22% of SCCs harbour oncogenic mutations in RAS (9% HRAS, 8% NRAS, 5% KRAS: www.sanger.ac.uk/genetics/CGP/cosmic/), raising the intriguing possibility that the BRAF-selective drugs act as tumor promoters in premalignant skin cells harboring existing mutations in RAS and/or activation of upstream components that activate RAS.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	While sorafenib is equipotent for wild-type and BRAF (Wilhelm et al., 2004), the BRAF inhibitors we used are approximately 10-fold more active against BRAF (King et al., 2006; Tsai et al., 2008).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Nevertheless, our data establish that they target wild-type BRAF in RAS mutant cells.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The problem of mutant v.s. wild-type protein specificity is likely to be difficult to resolve, because whereas full inhibition of BRAF may be necessary for clinical response in BRAF mutant tumors, activation of only a small proportion of wild-type BRAF could be sufficient to activate the pathway in RAS mutant cells.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Thus, to achieve efficacy against BRAF but avoid activation of wild-type BRAF in RAS mutant cells, the drugs will need to be exquisitely selective for the mutant protein.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Alternatively, pan-RAF drugs may be effective because they will target both BRAF and CRAF activated by BRAF in RAS mutant tumors.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Furthermore, our data suggest that CRAF or MEK selective drugs should be used in RAS mutant tumors, because they do not induce BRAF-CRAF complexes and will not activate the pathway if the tumors acquire mutations such as CRAF that block drug binding.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Perhaps RAF and MEK inhibitors should be combined to provide the best responses and prevent emergence of resistance, but these issues need to be balanced against the urgency of the clinical problem being addressed.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	In summary, we show that inhibition of BRAF in RAS mutant cancer cells leads to MEK hyperactivation through CRAF.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	We have elucidated another mechanism by which BRAF activates MEK–ERK signaling, not only to drive tumorigenesis and tumor progression, but also potentially to allow development of de novo or acquired resistance to RAF-targeted therapies.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Clearly, BRAF is a remarkably versatile oncogene that can promote MEK–ERK activation and tumor progression through several mechanisms and these will require different therapeutic strategies for effective disease management.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Notably, many of the mutations that occur in other kinases in cancer are also predicted to cause inactivation (www.sanger.ac.uk/genetics/CGP/cosmic/).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Our data raise the possibility that these could also act as idiosyncratic gain-of-function mutations that drive tumorigenesis.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	This study also raises important clinical questions and highlights the importance of fully understanding how signaling networks function to fully comprehend how patients may respond to targeted drugs.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	They also highlight the importance of genetic screening for patients, not only to identify those who are likely to respond, but to exclude those who could experience adverse effects and thereby ensure successful implementation of personalized medicine.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Expression vectors for epitope-tagged BRAF and CRAF have been described (Wan et al., 2004).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For western blotting the following antibodies were used: rabbit anti-ppMEK1/2 and mouse anti-myc 9B11 (Cell Signaling Technology); mouse anti-NRAS (C-20), rabbit anti-ERK2 (C-14), rabbit anti-ARAF (C-20), mouse anti-BRAF (F-7) (Santa Cruz Biotechnology); mouse anti-Tubulin, and mouse anti-ppERK1/2 (Sigma); mouse anti-CRAF (for western blotting) (BD Transduction Laboratories).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For immunoprecipitation, the following antibodies were used: rabbit anti-myc (Abcam); rabbit anti-CRAF (C-20;Santa Cruz Biotechnology); mouse anti-BRAF (F-7) (Ab from Santa Cruz Biotechnology).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Calf intestinal phosphatase (CIP) was from New England Biolabs (NEB).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	PD184352, sorafenib and PLX4720 were synthesized in-house; 885-A was synthesized by Evotec AG (Abingdon, UK).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	All drugs were prepared in DMSO.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Synthetic routes are available on request.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Human cell lines were cultured in DMEM (A375, WM852, HCT116, SW620, and PMWK) or RPMI (D04, MM485, MM415, and WM1791c) supplemented with 10% fetal bovine serum.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For protein depletion, 3 × 10 D04 cells were transfected with 5nM CRAF (5′-AAGCACGCTTAGATTGGAATA-3′) or NRAS (5′-CATGGCACTGTACTCTTCTCG-3′) specific, or scrambled control (5′-AAACCGTCGATTTCACCCGGG-3′) siRNA using INTERFERin as recommended by the manufacturer (Polyplus Transfection SA).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For transient expression studies, D04 cells were transfected using the Amaxa Nucleofector System as recommended by the manufacturer (Lonza).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	COS-7 cells were propagated, transfected, and extracted as described (Wan et al., 2004).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For generation of stable lines, D04 cells were transfected with pMCEF-FLAG-CRAF or pMCEF-FLAG-CRAF using Effectene as recommended by the manufacturer (Invitrogen) and selected in G418 (1 mg/ml).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Cell lysates were prepared with NP40 buffer as described (Wan et al., 2004).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For immunoprecipitation, lysates were incubated with 2 μg BRAF F-7, 5 μg CRAF C-20 or 2 μg rabbit anti-myc antibodies, captured on Protein G sepharose 4B beads (Sigma) and analyzed by western blotting using standard protocols.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Specific bands were detected using fluorescent-labeled secondary antibodies (Invitrogen; Li-COR Biosciences) and analyzed using an Odyssey Infrared Scanner (Li-COR Biosciences).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For CIP treatment, immunoprecipitates were washed twice with NP40 lysis buffer, once in CIP buffer (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 10 mM MgCl2, and 1 mM EDTA), and incubated with CIP with or without 0.2 mM Na3VO4 and 7 mM EDTA.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The immunoprecipitates were washed in CIP buffer and western blotted.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Coupled RAF kinase assays were performed with immunoprecipitated CRAF or BRAF as described (Wan et al., 2004).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Membrane fractionation was as described (Garnett et al., 2005).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Experiments were performed under Home Office license authority in accordance with United Kingdom Coordinating Committee on Cancer Research Guidelines (Workman et al., 1988) and with local Ethics Committee approval.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	To activate CreERT2, mice were treated with four doses (10mg each) of topically applied tamoxifen as described (Dhomen et al., 2009).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Genotyping was performed by PCR.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Braf and Braf was analyzed as described for Braf and Braf respectively and Tyr::CreERT2 was analyzed as described (Dhomen et al., 2009).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Kras was analyzed using primers 5′-CGCAGACTGTAGAGCAGCG-3′ and 5′-CCATGGCTTGAGTAAGTCTGC-3′. For expression analysis, RNA was prepared (QIAGEN RNEasy, QIAGEN) and first-strand cDNA synthesis was performed with 500ng total RNA and random hexanucleotides (Random Primers, Invitrogen).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Specific genes were amplified under linear conditions for analysis as described (Dhomen et al., 2009).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For Kras cDNA sequencing, a 238 bp fragment of Kras cDNA was PCR amplified using primers 5′-GGCGGCAGCGCTGTGGCGGCG-3′ and 5′-CGTAGGGTCATACTCATCCAC-3′ and sequenced using automated dideoxy sequencing.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For immunohistochemistry (IHC), tissues were fixed and analyzed as described (Dhomen et al., 2009).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Positive (a well characterized sample of mouse melanoma) and negative (omission of the primary antibody and substitution with preimmune serum) controls were included in each slide run.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Immunohistochemical staining was analyzed by two of the authors on a multi-headed microscope.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Tumor cell lines were established by mechanically dissociating tumors in DMEM/20%FCS/Primocin (0.1mg/ml - InvivoGen) and clonal lines were selected by limiting dilution.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

They were highly proliferative as evidenced by large numbers of mitotic figures in the superficial and deep aspects of the lesions (∼6 mitosis/10HPF; Figure 5H) and positive staining for Ki67 throughout (Figure 5I).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The tumors were strongly and diffusely positive for S100 (Figure 6A) and expressed the melanocyte markers tyrosinase, Dct, Pax3, and silver (Figure 6B), consistent with a diagnosis of melanoma.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Genomic DNA analysis of the tumors and cell lines derived from them confirmed that Braf had been recombined to Braf (Figure 6C).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

However, for technical reasons we could not detect Kras recombination (data not shown), so used RT-PCR to amplify and sequence Kras mRNA.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

We show that only wild-type Kras is expressed in the kidneys, whereas the tumors expressed both wild-type Kras and Kras (Figure 6D).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Importantly, we show constitutive binding of Braf to Craf in cells from the Kras/Braf tumors (Figure 6E).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

As a control, we used cells from melanoma induced by Kras overexpression.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Briefly, when Kras was overexpressed in melanocytes in mice using the β-actin promoter (β-actin:LSL:Kras; Meuwissen et al., 2001), it induced rapid onset melanoma (median time to onset 2 months, 100% penetrance within 3 months) in the absence of Braf (manuscript submitted).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Importantly, in cells from these tumors, Braf does not bind to Craf (Figure 6E).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Thus, it is only kinase-dead Braf and not wild-type Braf that binds to Craf in the presence of oncogenic Kras.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

In this study, we show that inhibition of BRAF by chemical or genetic means in the presence of oncogenic or growth-factor activated RAS induces BRAF binding to CRAF, leading to CRAF hyperactivation and consequently elevated MEK and ERK signaling.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The mechanism we describe is another paradigm of RAF activation downstream of RAS and based on our findings, we propose the following mechanism by which this occurs.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

We posit that in RAS mutant cells, BRAF maintains itself in an inactive conformation through its own kinase activity, either through auto-phosphorylation, or by phosphorylating a partner protein that then keeps it inactive (Figure 7A).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

We are currently using mass-spectrometry and mutagenic approaches to elucidate the underlying mechanism.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

We propose that when BRAF is inhibited, it escapes this auto-inhibited state and is recruited to the plasma membrane by RAS, where it forms a stable complex with CRAF.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Critically, we posit that because it is inhibited, BRAF does not directly phosphorylate MEK, but rather it acts as a scaffold whose function is to enhance CRAF activation, thereby allowing CRAF to hyperactivate the pathway (Figure 7B).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

We do not know the stoichiometry of the components in these complexes, but since BRAF and CRAF must both bind to RAS for complex formation, it seems likely that at least two RAS proteins are needed to stimulate formation of the complex (Figure 7B).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

It is unclear why PLX4720 only induces weak binding of BRAF to CRAF, but this may stem from its unique property of displacing the α-C helix of BRAF when it binds (Tsai et al., 2008) and suggests that this helix is important for BRAF binding to CRAF, something that will only be resolved when the BRAF:CRAF crystal structure is solved.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

We have attempted to identify other proteins that may be required to stabilize the BRAF–CRAF complexes.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Our unpublished mutagenesis data suggests that 14-3-3 is required to stabilize these drug-induced complexes (data not shown) and this is consistent with previous observations demonstrating that 14-3-3 mediates BRAF binding to CRAF (Garnett et al., 2005; Rushworth et al., 2006).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Although this appears to contradict our observation that dephosphorylation does not disrupt the complex, because 14-3-3 binds to BRAF and CRAF in a phosphorylation-dependent manner, we presume that 14-3-3 protects these sites from dephosphorylation.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

We have also used RNAi to examine the potential role of other proteins implicated in BRAF-CRAF complex formation or pathway activation, including the scaffold proteins KSR, Sprouty2 and RKTG and the small G protein RHEB, but our preliminary results have not revealed obvious roles for these proteins.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Our studies have parallels to the recently described heterodimers between DRAF and KSR in Drosophila (Rajakulendran et al., 2009).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Notably, flies have only one RAF isoform and it appears to be an ortholog of BRAF rather than ARAF or CRAF.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Our inability to demonstrate an obvious role for KSR in mediating BRAF binding to CRAF or CRAF activation by BRAF suggests that the mechanism underlying dimerization here may be different from those described in flies, but clearly additional studies are required to investigate further the role of scaffold proteins in mediating the phenomena we report.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

In contrast to the BRAF-selective inhibitors, the pan-RAF inhibitors appear to induce paradoxical activation of CRAF.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

They induce BRAF binding to CRAF and CRAF activation, but do not activate MEK–ERK signaling.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

We posit that this is because these agents target both BRAF and CRAF.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Thus, although their inhibition of BRAF will stimulate CRAF activation, they will simultaneously inhibit CRAF (Figure 7C).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

This model is supported by our observation that CRAF converts sorafenib from a pathway inhibitor to a pathway activator and we argue that the paradoxical activation of CRAF by these inhibitors is mediated by BRAF, rather than disrupted feedback inhibition as previously suggested (Hall-Jackson et al., 1999).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Recently, paradoxical activation of PKB/AKT and PKCɛ was also described (Cameron et al., 2009; Okuzumi et al., 2009).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

While ATP-competitive inhibition can block kinase function, they do not block the upstream events that activate the target kinase.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For instance, PKB/AKT inhibitors block the function of this kinase, but occupation of the ATP-pocket by these inhibitors was sufficient to induce the priming phosphorylation usually required for its full activation (Okuzumi et al., 2009).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Inhibitor binding to PKCɛ has been shown to have a similar effect (Cameron et al., 2009).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Importantly, the paradoxical activation of PKB/AKT and PKCɛ did not result in pathway activation because of the continued presence of the inhibitors (Frye and Johnson, 2009).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

In contrast, although BRAF inhibitors also block BRAF kinase activity, this relieves auto-inhibition and results in BRAF hyperphosphorylation, BRAF binding to CRAF, pathway activation and oncogenesis, all presumably because BRAF can heterodimerize with CRAF.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Our study also highlights the critical difference between BRAF-selective and pan-RAF drugs.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Whereas BRAF-selective drugs cause pathway activation in a RAS-dependent manner, this does not occur with pan-RAF drugs.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Our results provide important insight into the genetics of human cancer.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Excluding V600 mutants, D594 mutants are the third most common in BRAF in cancer (34 out of 443 cases or ∼7.7%; www.sanger.ac.uk/genetics/CGP/cosmic/).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Furthermore, as mentioned in the Introduction, while BRAF and RAS mutations are generally mutually exclusive, 4 of the 34 (11.8%) tumors with D594 mutations also have mutations in RAS.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

This is a highly significant enrichment for the coincidence of these mutations (p < 10; Fisher's Exact Test) and suggests a functional interaction.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

We now provide strong circumstantial evidence of such an interaction using transgenic mice.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

By themselves, Braf and Kras do not induce melanoma, but they cooperate to induce rapid onset melanoma.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

This highly significant result (p < 0.0002) provides a rational explanation for the coincidence of these mutations in human cancer.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Furthermore, we show that the BRAF inhibitors also hyperactivate this pathway in growth factor stimulated cells, providing an explanation of why kinase dead BRAF mutations are not always coincident with RAS mutations; presumably in some tumors the cooperating mutation is upstream of RAS.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Our results also suggest several potential mechanisms by which resistance to RAF targeting drugs could develop in patients.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

BRAF mutant tumors could become resistant to BRAF-selective drugs, if they acquire a mutation in RAS or an upstream component that activates RAS, or if the drugs select a population of cells harboring pre-existing mutations in RAS.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Theoretically this would cause BRAF-mediated CRAF activation, which may not only induce resistance, but could potentially promote tumor growth.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

In line with this, increased expression of CRAF can mediate acquired resistance to pan-RAF drugs in BRAF mutant cancer cells in vitro (Montagut et al., 2008), establishing that CRAF can mediate resistance under some circumstances.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Our in vitro studies also suggest that a potential mechanism of resistance in patients with RAS mutant tumors being treated with pan-RAF drugs is acquisition (or selection for cells with pre-existing mutations) of a CRAF mutation such as a gatekeeper mutant that prevents drug binding.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Again this would potentially result in BRAF-mediated activation of CRAF (Figure 7D) and possibly accelerated tumor growth.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Although our studies are restricted to cell lines and transgenic mice, they do have important immediate clinical implications.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

They strongly argue that BRAF-selective inhibitors should not be administered to patients with RAS mutant tumors, because long-term use could accelerate tumor growth.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Intriguingly, 10%–15% of patients treated with BRAF-selective drugs develop squamous cell carcinoma (SCC)(Flaherty et al., 2009; Schwartz et al., 2009).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Although MEK–ERK signaling has not yet been implicated in this response, 22% of SCCs harbour oncogenic mutations in RAS (9% HRAS, 8% NRAS, 5% KRAS: www.sanger.ac.uk/genetics/CGP/cosmic/), raising the intriguing possibility that the BRAF-selective drugs act as tumor promoters in premalignant skin cells harboring existing mutations in RAS and/or activation of upstream components that activate RAS.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

While sorafenib is equipotent for wild-type and BRAF (Wilhelm et al., 2004), the BRAF inhibitors we used are approximately 10-fold more active against BRAF (King et al., 2006; Tsai et al., 2008).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Nevertheless, our data establish that they target wild-type BRAF in RAS mutant cells.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The problem of mutant v.s. wild-type protein specificity is likely to be difficult to resolve, because whereas full inhibition of BRAF may be necessary for clinical response in BRAF mutant tumors, activation of only a small proportion of wild-type BRAF could be sufficient to activate the pathway in RAS mutant cells.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Thus, to achieve efficacy against BRAF but avoid activation of wild-type BRAF in RAS mutant cells, the drugs will need to be exquisitely selective for the mutant protein.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Alternatively, pan-RAF drugs may be effective because they will target both BRAF and CRAF activated by BRAF in RAS mutant tumors.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Furthermore, our data suggest that CRAF or MEK selective drugs should be used in RAS mutant tumors, because they do not induce BRAF-CRAF complexes and will not activate the pathway if the tumors acquire mutations such as CRAF that block drug binding.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Perhaps RAF and MEK inhibitors should be combined to provide the best responses and prevent emergence of resistance, but these issues need to be balanced against the urgency of the clinical problem being addressed.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

In summary, we show that inhibition of BRAF in RAS mutant cancer cells leads to MEK hyperactivation through CRAF.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

We have elucidated another mechanism by which BRAF activates MEK–ERK signaling, not only to drive tumorigenesis and tumor progression, but also potentially to allow development of de novo or acquired resistance to RAF-targeted therapies.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Clearly, BRAF is a remarkably versatile oncogene that can promote MEK–ERK activation and tumor progression through several mechanisms and these will require different therapeutic strategies for effective disease management.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Notably, many of the mutations that occur in other kinases in cancer are also predicted to cause inactivation (www.sanger.ac.uk/genetics/CGP/cosmic/).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Our data raise the possibility that these could also act as idiosyncratic gain-of-function mutations that drive tumorigenesis.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

This study also raises important clinical questions and highlights the importance of fully understanding how signaling networks function to fully comprehend how patients may respond to targeted drugs.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

They also highlight the importance of genetic screening for patients, not only to identify those who are likely to respond, but to exclude those who could experience adverse effects and thereby ensure successful implementation of personalized medicine.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Expression vectors for epitope-tagged BRAF and CRAF have been described (Wan et al., 2004).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For western blotting the following antibodies were used: rabbit anti-ppMEK1/2 and mouse anti-myc 9B11 (Cell Signaling Technology); mouse anti-NRAS (C-20), rabbit anti-ERK2 (C-14), rabbit anti-ARAF (C-20), mouse anti-BRAF (F-7) (Santa Cruz Biotechnology); mouse anti-Tubulin, and mouse anti-ppERK1/2 (Sigma); mouse anti-CRAF (for western blotting) (BD Transduction Laboratories).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For immunoprecipitation, the following antibodies were used: rabbit anti-myc (Abcam); rabbit anti-CRAF (C-20;Santa Cruz Biotechnology); mouse anti-BRAF (F-7) (Ab from Santa Cruz Biotechnology).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Calf intestinal phosphatase (CIP) was from New England Biolabs (NEB).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

PD184352, sorafenib and PLX4720 were synthesized in-house; 885-A was synthesized by Evotec AG (Abingdon, UK).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

All drugs were prepared in DMSO.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Synthetic routes are available on request.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Human cell lines were cultured in DMEM (A375, WM852, HCT116, SW620, and PMWK) or RPMI (D04, MM485, MM415, and WM1791c) supplemented with 10% fetal bovine serum.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For protein depletion, 3 × 10 D04 cells were transfected with 5nM CRAF (5′-AAGCACGCTTAGATTGGAATA-3′) or NRAS (5′-CATGGCACTGTACTCTTCTCG-3′) specific, or scrambled control (5′-AAACCGTCGATTTCACCCGGG-3′) siRNA using INTERFERin as recommended by the manufacturer (Polyplus Transfection SA).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For transient expression studies, D04 cells were transfected using the Amaxa Nucleofector System as recommended by the manufacturer (Lonza).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

COS-7 cells were propagated, transfected, and extracted as described (Wan et al., 2004).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For generation of stable lines, D04 cells were transfected with pMCEF-FLAG-CRAF or pMCEF-FLAG-CRAF using Effectene as recommended by the manufacturer (Invitrogen) and selected in G418 (1 mg/ml).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Cell lysates were prepared with NP40 buffer as described (Wan et al., 2004).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For immunoprecipitation, lysates were incubated with 2 μg BRAF F-7, 5 μg CRAF C-20 or 2 μg rabbit anti-myc antibodies, captured on Protein G sepharose 4B beads (Sigma) and analyzed by western blotting using standard protocols.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Specific bands were detected using fluorescent-labeled secondary antibodies (Invitrogen; Li-COR Biosciences) and analyzed using an Odyssey Infrared Scanner (Li-COR Biosciences).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For CIP treatment, immunoprecipitates were washed twice with NP40 lysis buffer, once in CIP buffer (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 10 mM MgCl2, and 1 mM EDTA), and incubated with CIP with or without 0.2 mM Na3VO4 and 7 mM EDTA.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The immunoprecipitates were washed in CIP buffer and western blotted.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Coupled RAF kinase assays were performed with immunoprecipitated CRAF or BRAF as described (Wan et al., 2004).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Membrane fractionation was as described (Garnett et al., 2005).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Experiments were performed under Home Office license authority in accordance with United Kingdom Coordinating Committee on Cancer Research Guidelines (Workman et al., 1988) and with local Ethics Committee approval.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

To activate CreERT2, mice were treated with four doses (10mg each) of topically applied tamoxifen as described (Dhomen et al., 2009).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Genotyping was performed by PCR.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Braf and Braf was analyzed as described for Braf and Braf respectively and Tyr::CreERT2 was analyzed as described (Dhomen et al., 2009).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Kras was analyzed using primers 5′-CGCAGACTGTAGAGCAGCG-3′ and 5′-CCATGGCTTGAGTAAGTCTGC-3′. For expression analysis, RNA was prepared (QIAGEN RNEasy, QIAGEN) and first-strand cDNA synthesis was performed with 500ng total RNA and random hexanucleotides (Random Primers, Invitrogen).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Specific genes were amplified under linear conditions for analysis as described (Dhomen et al., 2009).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For Kras cDNA sequencing, a 238 bp fragment of Kras cDNA was PCR amplified using primers 5′-GGCGGCAGCGCTGTGGCGGCG-3′ and 5′-CGTAGGGTCATACTCATCCAC-3′ and sequenced using automated dideoxy sequencing.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For immunohistochemistry (IHC), tissues were fixed and analyzed as described (Dhomen et al., 2009).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Positive (a well characterized sample of mouse melanoma) and negative (omission of the primary antibody and substitution with preimmune serum) controls were included in each slide run.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Immunohistochemical staining was analyzed by two of the authors on a multi-headed microscope.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Tumor cell lines were established by mechanically dissociating tumors in DMEM/20%FCS/Primocin (0.1mg/ml - InvivoGen) and clonal lines were selected by limiting dilution.