Yanping Li, Maho Takahashi, and Philip J. S. Stork 1 From the Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239

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1 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 38, pp , September 20, by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Ras-mutant Cancer Cells Display B-Raf Binding to Ras That Activates Extracellular Signal-regulated Kinase and Is Inhibited by Protein Kinase A Phosphorylation * Received for publication, February 19, 2013, and in revised form, July 22, 2013 Published, JBC Papers in Press, July 26, 2013, DOI /jbc.M Yanping Li, Maho Takahashi, and Philip J. S. Stork 1 From the Vollum Institute, Oregon Health and Science University, Portland, Oregon Background: How the camp-dependent protein kinase PKA regulates B-Raf binding to Ras is not known. Results: PKA inhibits the binding of B-Raf to active Ras via phosphorylation of serine 365 in B-Raf. Conclusion: B-Raf can participate in the Ras-dependent activation of ERK in Ras-mutant cancers, and this is inhibited by PKA. Significance: PKA can block Ras binding to both Raf isoforms C-Raf and B-Raf. The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the camp-dependent protein kinase PKA. camp inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that camp and hormones coupled to camp may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf. Ras-dependent activation of ERKs triggers cell proliferation in both normal and malignant cells. Many human tumors display constitutive activation of the MAP kinase cascade that dictates their high proliferation rate. This activation is commonly achieved by mutations in the family of Ras GTPase that maintain Ras in a basally active (GTP-loaded) state. Indeed, oncogenic Ras mutations are present in nearly 30% of human tumors * This work was supported, in whole or in part, by National Institutes of Health Grant DK (to P. J. S. S.). This work was also supported by the American Association of Cancer Research Grant AACR (to Y. L.). 1 To whom correspondence should be addressed: Vollum Institute, Oregon Health and Science University, L474, 3181 SW Sam Jackson Park Rd., Portland, OR Tel.: ; Fax: ; stork@ ohsu.edu. (1, 2). Not surprisingly, blocking the MAP kinase (ERK) cascade downstream of Ras is a well-explored therapeutic strategy (3 5). For example, the MAP kinase kinase kinase B-Raf is a validated target in human melanomas harboring mutant B-Raf oncogenes (6, 7). Interestingly, the use of some Raf inhibitors is contraindicated in patients with melanomas harboring oncogenic Ras mutations (6, 7). Melanoma cells with oncogenic Ras mutations have provided additional insights into the roles of Raf isoforms in human tumors. For example, melanoma cells with oncogenic Ras mutations undergo a switch in the usage of Raf isoforms from B-Raf to C-Raf (8). This novel dependence on C-Raf occurs despite the continued expression of B-Raf within the tumor cells (9). Both the proliferation and basal ERK activation in these Ras-mutant melanoma cells were dependent solely on C-Raf, with no contribution from B-Raf. This was due to the high basal level of ERK activity in these cancer cells and the subsequent ERK-dependent phosphorylation of B-Raf itself, which prevented B-Raf association with oncogenic Ras (9, 10). Whether this isoform switch from B-Raf to C-Raf occurs in non-melanocytic tumors that harbor oncogenic Ras mutations is unknown. To determine whether B-Raf contributes to the malignant phenotype of non-melanocytic cancer cells will be important because of the potential of B-Raf-specific therapies and the possibility that signaling from Ras to C-Raf and B-Raf may be differentially inhibited by PKA. Oncogenic Ras mutations are prevalent in lung and colon cancers (11 13). Ras mutations occur in 15 30% of non-small cell lung carcinomas (NSCLC) 2 and are associated with a poor prognosis (14). Most Ras mutations arise in either the Kirsten Ras (KRas) or neuroblastoma Ras (NRas) isoforms. NRas and KRas are common in both lung and colon cancers and are thought to induce carcinogenesis through similar functional outputs (13). However, differences between NRas and KRas have been noted in mouse models of colonic cancer (15). 2 The abbreviations used are: NSCLC, non-small cell lung carcinoma; NRas, neuroblastoma RAS viral (v-ras) oncogene homolog; F/I, forskolin/3-isobutyl-1-methylxanthine; IP, immunoprecipitation; GTP S, guanosine 5-3-O- (thio)triphosphate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Ab, antibody JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 NUMBER 38 SEPTEMBER 20, 2013

2 These Ras-mutant tumors generally express wild type B-Raf (11). Therefore, it is important to revisit the requirement of B-Raf in this group of Ras-mutant cells. Here we examined a range of Ras-mutant cancer cell lines including H1299 cells, a human NSCLC line, harboring an NRas Q61K mutation, Calu-6, a human NSCLC line (KRas Q61K), and HCT116, a human colorectal carcinoma cell line (KRas G13D). Although these cells generally express B-Raf, the contribution of B-Raf to oncogenic Ras signaling is not known. We show that both B-Raf and C-Raf contribute to the basal ERK activation seen in these cells. Moreover, both C-Raf and B-Raf were found to be constitutively associated with oncogenic Ras and that the binding of each isoform was inhibited through independent phosphorylations of each kinase by PKA. EXPERIMENTAL PROCEDURES Reagents and Buffers Forskolin, 3-isobutyl-1-methylxanthine, and UO126 were purchased from Calbiochem. Phorbol- 12-myristate-13-acetate, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), GTP, GTP S, glutathione peptide, and glutathione-agarose beads were from Sigma. PACAP38 was from Bachem (Torrance, CA). Lysis buffer contained 50 mm Tris/HCl (ph 7.8), 1% Nonidet P-40, 10% glycerol, 200 mm NaCl, 2 mm MgCl 2, 0.5 mm -glycerol phosphate, protease, and phosphatases inhibitors: 1 mm phenylmethylsulfonyl fluoride, 2 g/ml vanadate, 10 g/ml trypsin inhibitor, 1 g/ml leupeptin, 2 g/ml aprotinin, and 5 mm NaF. Plasmids and Antibodies The B-Raf and C-Raf plasmids have been previously described (16). FLAG-NRas constructs were generated from human NRas provided by the Missouri S&T cdna Resource Center (Rolla, MO). The Myc-C-Raf S43A, Myc-C-Raf S259A, HA-B-Raf S365A, HA-B-Raf S151A, HA-B-Raf R509H, GFP-B-Raf R188L, and GFP-RapE63 mutants were generated by site-directed mutagenesis using the QuikChange TM kit (Stratagene, La Jolla, CA). Myc was used for all experiments examining and was provided by Gary Thomas (University of Pittsburgh, Pittsburgh, PA). Protein kinase inhibitor (of PKA) PKI was provided by Richard Maurer (Oregon Health and Science University, Portland, OR). Complementary pairs of short hairpin RNA (shrna) oligonucleotides were synthesized by Integrated DNA Technologies Inc. (Coralville, IA), annealed, and cloned into the psuper-neo GFP vector (OligoEngine, Seattle, WA). The sense shrna sequences oligonucleotides were: C-Raf shrna, CATCAGACAACTCT- TATTG (17); B-Raf shrna, ACAGAGACCTCAAGAGTAA (18); NRas shrna1, ATACGCCAGTACCGAATGA (19); and NRas shrna2, CAGCAGTGATGATGGGACT (20). Human H-RasV12 (RasV12) in pgex-4t3 was previously described (21). B-Raf constructs for bacterial expression were generated by PCR amplification. PCR primers containing Sac1 and HindIII sites were designed to amplify DNA encoding amino acids of human B-Raf WT or R188L mutant. The PCR products were subcloned into the pet24a vector (Millipore, Inc.) to generate amino-terminal fragments containing the Ras binding domains of B-Raf WT or R188L. Anti-ERK2 (D2), anti-b-raf (H145), anti-b-raf (F7), anti-c- Raf (C12), anti-nras (C20), anti-gfp, and anti-c-myc (9E10) antibodies and anti-c-myc (9E10) beads were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-pan-Ras (Ras10) was from EMD Millipore (Billerica, MA). Anti-C-Raf monoclonal antibody was from BD Transduction Laboratories. Anti-FLAG antibody and beads were purchased from Sigma. Anti-phospho-ERK (T201 and Y204) (Ab #9101), Anti-phospho- MEK (S217/221) (Ab #9154S), anti-phospho-c-raf (Ser(P)- 259) (Ab #9421), and anti-phospho-mapk/cdk substrate antibody (Ab #2325) were from Cell Signaling Technology (Beverly, MA). Anti-HA (clone 16B12) antibody was from Covance Inc (Princeton, NJ). Cell Culture Conditions and Transfections H1299 cells were provided by Mushui Dai (Oregon Health and Science University). HCT116 cells were provided by Charles Lopez (Oregon Health and Science University). Calu-6 cells were purchased from American Type Culture Collection (Manassas, VA). H1299, HCT116, Calu-6, and Hek293 cells were cultured in Dulbecco s modified Eagle s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin, and L-glutamine at 37 C in 5% CO 2. Beas-2b cells were a gift of David Jacoby (Oregon Health and Science University) and were cultured serum-free in 50% DMEM and 50% F-12 medium supplemented with penicillin, streptomycin, and L-glutamine at 37 C in 5% CO 2. Before treatment, cells were serum-starved in medium with 0.2% FBS overnight and then treated with 10 M forskolin and 100 M 3-isobutyl-1-methylxanthine for 10 min or the indicated times. PACAP38 was used at 500 nm for 10 min. EGF and PDGF were used at 50 ng/ml for 5 min unless otherwise indicated. Phorbol-12-myristate-13-acetate was used at 50 ng/ml. Cross-linking anti-cd3 antibodies were used as described (22). Immunoprecipitations and Western blotting were performed as previously described (23, 24). For all transfections, cells were transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen), and serum was starved before treatment. For shrna experiments, cells were transfected twice on consecutive days and serum-starved overnight. In all cases, representative Western blots of at least three independent experiments are shown. When shown, statistical significant was evaluated using an unpaired t test. MTT Proliferation Assay H1299 and HCT116 cells were plated at 2,500 or 20,000 cells per well in 96-well plates, respectively. 6 8 wells were used for each condition. After 24 h, cells were serum-starved and treated with F/I and UO126 (10 M). After 3 days, cell density was assessed by an MTT assay, as per the manufacturer s instructions. The absorbance was read at 590 nm using SpectraMax M2 microplate reader. For each cell line, a standard curve was generated to establish a linear range of ODs versus cell number. Relative cell numbers are presented as the percent of cell numbers in the untreated condition. The averages of three independent experiments are shown, and statistical significant was evaluated using an unpaired t test. ERK Phosphorylation in Vitro The ERK2 phosphorylation reaction was carried out in vitro after B-Raf immunoprecipitation previously described (21 23). The immunoprecipitation complex was washed, resuspended in kinase reaction buffer (50 mm Tris/HCl (ph 7.5), 0.02 mm EGTA, 2 g/ml vanadate, 5 mm NaF, 1 M DTT), and incubated with recombinant active ERK2 (20 ng, EMD Millipore; catalog #14-550M) and ATP (1 mm in SEPTEMBER 20, 2013 VOLUME 288 NUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 27647

3 FIGURE 1. Both B-Raf and C-Raf contribute to the basal ERK activation in Ras-mutant cancer cells. A, basal ERK activation in H1299 cells is dependent on NRas. H1299 cells were transfected with one of two shrnas for NRas (shrna1, shrna2) or control shrna. Cells were harvested after 48 h, and ERK activation was measured by Western blot using phosphorylation-specific antibodies against phosphorylated/activated ERK (perk) (first panel). Total endogenous ERKs are shown as a loading control in the second panel. The efficiencies of knockdown of endogenous NRas by each shrna are shown in the third panel. B, the expression levels of endogenous B-Raf (first panel) and C-Raf (second panel) in three Ras-mutant cancer cells (HCT116, H1299, and Calu-6 cells) are shown in the left, middle, and right lanes, respectively. Total endogenous ERK2 levels are shown in the third panel as a loading control. C E, both B-Raf and C-Raf contribute to the basal ERK activation in H1299 cells (C), HCT116 cells (D), and Calu-6 cells (E). Cells were transfected with control shrna, B-Raf shrna, or C-Raf shrna as indicated. Cells were harvested, and endogenous protein levels were assayed by Western blot. The efficiencies of knockdown of endogenous B-Raf and C-Raf are shown in the first and second panels, respectively. MEK activation was measured using phosphorylation-specific antibodies against pmek (third panel). ERK activation was measured using phosphorylation-specific antibodies against perk (fourth panel). Total endogenous ERK2 levels are shown in the fifth panel. 10 mm MgCl 2 ) and incubated at 30 ºC for 30 min. Proteins were eluted and detected by immunoblotting with the indicated antibodies. Protein Purification and Expression The plasmids encoding proteins for bacterial expression were transformed into bacterial strain BL21(DE3). Expression of His-BRaf (amino acids 1 414) and His-BRaf R188L (amino acids 1 414) were induced by1mm isopropyl- -D-thiogalactopyranoside at 37 C for 4 h after an A 600 of 0.6 was reached. The proteins were purified as described (24). RasV12 was expressed and purified as a GST fusion protein, and RasV12 was released after cleavage of the GST peptide, as described previously (21). In Vitro Binding Assay 100 ng of RasV12 per tube was loaded with 0.1 mm GTP S or1mm GDP in 200 l of lysis buffer supplemented with 10 mm EDTA at 30 C for 30 min as described (24). The reaction was stopped by the addition of MgCl 2 at 10 mm. 1 g of His-BRaf (amino acids 1 414) wild type or R188L was incubated with RasV12-GTP S or RasV12- GDP in the presence of 20 l of His beads (50% slurry) for1hat 4 C. The beads were washed 3 times with 1 ml of lysis buffer, boiled in 1 SDS loading dye, and subjected to Western blotting. Preparation of Primary T Cells C57/BL6 mice were used to prepare splenic lymphocytes. Experiments on animals were performed according to the ethical guidelines of the Institutional Animal Care and Use Committee at Oregon Health and Science University in accordance with federal regulations regarding approved animal use and care. Cross-linking anti- CD3 antibodies were used as described (22). RESULTS Both B-Raf and C-Raf Are Required for Basal ERK Activation in H1299 Cancer Cells Cancer cells harboring Ras mutations maintain a high basal level of ERK activity that drives their proliferation (13). Moreover, Ras-mutant tumors remain dependent on Ras activation for ERK activation and proliferation (25). This was seen in H1299 cells, as basal ERK activation was blocked by shrnas for NRas (Fig. 1A). Ras activation of ERKs requires the action of Raf kinases. H1299, HCT116, and Calu-6 cells all express both B-Raf and C-Raf (Fig. 1B). Importantly, both B-Raf and C-Raf contributed to the basal ERK activity in all cells, as isoform-specific shrnas to B-Raf and C-Raf each reduced endogenous basal ERK activity (Fig. 1, C, D, and E). Similar reductions were seen in the basal activation of the ERK kinase MEK (Fig. 1, C, D, and E). Constitutive Ras Activation Does Not Prevent B-Raf Binding to Ras The role of B-Raf in maintaining the elevated ERK activity present in H1299 cells was surprising in light of the recent report that B-Raf is incapable of binding to oncogenic Ras in Ras-mutant melanomas (9, 10). The inability of B-Raf to bind to Ras in those tumors is a consequence of the ERKdependent phosphorylation of B-Raf on serine 151. Phosphorylation of the Ser-151 site disrupts the Ras binding domain of B-Raf (10, 26). The high level of basal ERK activation in Ras-mutant melanomas is thought to maintain this Ser-151 site in a constitutively phosphorylated state that directly interferes with the Ras-dependent activation of B-Raf signaling (10) JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 NUMBER 38 SEPTEMBER 20, 2013

4 FIGURE 2. The serine 151 site in B-Raf is largely unphosphorylated in H1299 cells. A, MAPK substrate antibody is selective for phosphorylation of Ser-151 on B-Raf. Cells were transfected with FLAG-B-Raf wild type (WT) or the FLAG-B-Raf S151A mutant (mut) as indicated. Cells were harvested, and FLAG-B-Raf was immunoprecipitated and incubated in kinase buffer in the absence ( ) or presence ( ) of active recombinant ERK2. The phosphorylations of both WT and mutant B-Raf were examined by Western blotting with the MAP kinase substrate antibody (MAPK substrate antibody (top panel). The levels of FLAG-B-Raf (WT or mut) within the immunoprecipitates are shown in the bottom panel. B, Ser-151 was largely unphosphorylated in H1299 cells. Cells were lysed, and endogenous B-Raf proteins were enriched by immunoprecipitation. Each sample was incubated in the absence ( ) or presence ( ) of active recombinant ERK2, and the Ser(P)-151 level was examined using the MAPK substrate Ab (top panel). The levels of endogenous B-Raf within the immunoprecipitates are shown in the bottom panel. C, B-Raf WT and B-Raf S151A bind equally well to FLAG-NRasV12. H1299 cells were transfected with HA-B-Raf wild type or HA-B-Raf S151A mutant in the presence or absence of FLAG-NRasV12, as indicated. Cells were harvested for IP with FLAG antibody, and the amount of HA-B-Raf (WT) or HA-B-Raf S151A (mut) protein bound to FLAG-NRasV12 was determined by Western blot using HA Ab (first panel). The levels of FLAG proteins within the FLAG IP are shown in the second panel, and the input levels of HA-B-Raf and FLAG-NRasV12 are shown in the third and fourth panels, respectively. D, phosphorylation of B-Raf on Ser-151 inhibits its association with active Ras. H1299 cells were transfected with either FLAG-B-Raf (first lane), HA-B-Raf WT and FLAG-NRasV12 (second lane), or HA-B-Raf S151A and FLAG-NRasV12 (third lane). All cells were subjected to IP using FLAG Ab (FLAG-B-Raf; first lane, FLAG-NRasV12; second and third lanes). The volumes of the protein loaded were adjusted to allow examination of equivalent amounts of B-Raf in each condition. The presence of Ser-151 phosphorylation within each IP was examined using the MAPK Substrate Ab (first panel). The third lane of this panel shows the control using B-Raf S151A, demonstrating the specificity of the MAPK substrate Ab for Ser(P)-151. The levels of B-Raf within each IP are shown using B-Raf Ab (second panel). The levels of FLAG-NRavV12 within each IP are shown in the third panel. Note that much less Ser(P)-151 was detected by the MAPK Substrate Ab in FLAG-NRavV12 IP (second lane) compared with the FLAG-B-Raf IP (first lane). Quantitation of binding was performed by ImageJ and normalized to total FLAG protein within the IP. The normalized data are represented as the mean S.E of four independent experiments. The bar graph shows the percentage of phosphorylated Ser-151, normalized for the level of B-Raf within each IP, compared with that seen in the first lane (100%). *, statistical significance is **, statistical significance is E, B-Raf WT and B-Raf S151E bind equally well to FLAG-NRasV12. H1299 cells were transfected with HA-B-Raf wild type or HA-B-Raf S151E mutant in the presence or absence of FLAG-NRasV12 as indicated. Cells were harvested for IP with FLAG antibody, and the amount of HA-B-Raf (WT) or HA-B-Raf S151E (mut) protein bound to FLAG-NRasV12 was determined by Western blot using HA Ab (first panel). The levels of FLAG proteins within the FLAG IP are shown in the second panel, and the input levels of HA-B-Raf (WT/mut) and FLAG-NRasV12 are shown in the third and fourth panels, respectively. Here, we assayed phosphorylation of this site using a MAP kinase substrate antibody that was designed to recognize a large subset of MAP kinase target sites. In H1299 cells, this antibody detected potential ERK-dependent phosphorylations in both transfected and endogenous B-Raf (Fig. 2, A and B), consistent with a basal level of ERK-dependent phosphorylation in B-Raf in these cells. B-Raf is known to be phosphorylated at multiple sites by ERK (26), yet this MAP kinase substrate antibody recognized only phosphorylation of Ser-151 (Ser(P)-151), as demonstrated by the absence of immunoreactivity of this MAP kinase substrate antibody with the mutant B-Raf S151A, both in vivo, and when B-Raf was phosphorylated by ERK2 in vitro (Fig. 2A). To establish the stoichiometry of phosphorylation of endogenous B-Raf Ser-151 in H1299 cells, we compared the basal levels of p Ser-151 in vivo to the levels of Ser(P)-151 achieved after in vitro phosphorylation of B-Raf by recombinant ERK2. The basal levels of Ser-151 phosphorylation of transfected (Fig. 2A) and endogenous B-Raf (Fig. 2 B) were greatly increased upon incubation with active ERK2 kinase in vitro (Fig. 2A and B), suggesting that Ser-151 was predominantly unphosphorylated in these tumor cells. An estimate of the stoichiometry of Ser-151 phosphorylation within cells could be made by comparing the Ser(P)-151 levels seen in cells with the maximal level achieved after the complete SEPTEMBER 20, 2013 VOLUME 288 NUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 27649

5 FIGURE 3. Basal B-Raf binding to active Ras is direct. A, the B-Raf R188L mutant prevents binding to RasV12 in vitro. The Ras binding domain of B-Raf (amino acids 1 414) and the corresponding R188L mutant were purified as His-tagged proteins and examined for their ability to bind GTP-loaded RasV12 in vitro. Proteins were purified and prepared as described under Experimental Procedures. Left panel, the bands corresponding to purified RasV12 (V12) and His-B-Raf wild type fragment (WT) and the His-B-Raf R188L fragment (RL) are shown by Coomassie staining after SDS-PAGE. Right panel, RasV12, loaded with either GTP S (first through third lanes) or GDP (fourth and fifth lanes), were incubated alone (first lane), with the His-tagged WT B-Raf fragment (second and fourth lanes), or with the RL mutant (third and fifth lanes) as indicated. The levels of His-B-Raf WT or RL fragments within the His pulldown assay are shown in the upper panel. The presence of RasV12 within the His pulldown assay is shown in the lower panel. B, the binding of B-Raf to NRasV12 is direct. H1299 cells were transfected with wild type GFP-B-Raf or the GFP-B-Raf R188L mutant in the presence or absence of FLAG-NRasV12 as indicated. The level of GFP-B-Raf associated with FLAG-NRasV12 is shown by Western blotting with GFP Ab after FLAG IP (first panel). The levels of FLAG proteins within the FLAG IP are shown in the second panel. The input levels of GFP proteins and FLAG-NRasV12 are shown in the third and fourth panels, respectively. C, binding of B-Raf to NRasV12 is independent of B-Raf dimerization. H1299 cells were transfected with wild type HA-B-Raf or the HA-B-Raf R509H mutant in the presence or absence of FLAG-NRasV12 as indicated. The level of HA-B-Raf (WT) or HA-B-Raf R509H (mut) associated with FLAG-NRasV12 was examined after FLAG IP by Western blotting with HA Ab (first panel). The FLAG-NRasV12 within the IP is also shown using Western blotting with FLAG Ab (second panel). The input levels of HA proteins (WT or mut) and FLAG-NRasV12 are shown (third and fourth panels). phosphorylation of B-Raf by active ERK2 kinase in vitro. In Fig. 2B, we show that incubation of immunoprecipitated B-Raf with active ERK kinase in vitro increased the basal level of phosphorylation of Ser fold over that seen in cells, suggesting that the stoichiometry of basal phosphorylation in vivo was not greater than 10 15%. Because in vitro phosphorylation can be incomplete, this may be an overestimate. The low level of basal phosphorylation of B-Raf Ser-151 was also suggested by the finding that wild type B-Raf and B-Raf S151A bound to NRasV12 to similar degrees (Fig. 2C). Therefore, in contrast to melanoma cells, the steady state level of phosphorylation at Ser-151 in these cells was not high enough to prevent a basal level of B-Raf binding to Ras. To determine whether phosphorylation on Ser-151 diminishes the ability of B-Raf to bind to Ras in H1299 cells, we compared the Ser(P)-151 levels with the entire pool of B-Raf to the pool of B-Raf bound to Ras. A significantly lower level of Ser(P)- 151 of B-Raf was detected within a Ras co-ip than in a B-Raf IP containing an equivalent amount of B-Raf protein (Fig. 2D), consistent with previous studies (10, 26). Interestingly, the phosphomimetic mutation B-Raf S151E maintained binding to Ras (Fig. 2E), suggesting that this mutation did not faithfully mimic phosphorylation. Taken together, the data suggest that the low level of phosphorylation of B-Raf on Ser-151 permits a high degree of basal B-Raf association with Ras in these cells. The basal association of B-Raf with Ras could be either direct or indirect through heterodimerization with C-Raf. Here, we examined whether the basal association of B-Raf with activated NRas in these cells was direct. For C-Raf, interaction with Ras in vitro can be blocked by a single change of arginine to leucine (R89L) (27). B-Raf R188L corresponds to this R89L mutation and, like C-Raf R89L, does not bind to Ras-GTP in vitro (Fig. 3A). In H1299 cells cotransfected with FLAG-NRasV12 and B-Raf R188L, B-Raf R188L was absent in NRas immunoprecipitates, confirming that the binding of B-Raf WT to Ras requires a direct interaction (Fig. 3B). The possibility that B-Raf was interacting with NRas through heterodimerization via C-Raf was also ruled out using the dimerization-deficient mutant B-Raf R509H (Fig. 3C). camp/pka Inhibits Basal ERK Activation and Proliferation in H1299 Cells Elevations in camp levels were triggered by the addition of forskolin, an activator of adenylate cyclase, in the presence of the phosphodiesterase inhibitor 3-isobutyl-1- methylxanthine (F/I). In H1299 cells, F/I blocked both basal MEK and ERK activity (Fig. 4A),as well as elevations in ERK activity triggered by EGF (Fig. 4B). F/I blocked basal ERK activation within 5 min (Fig. 4A). This block was also seen after incubation with the peptide hormone PACAP38 (pituitary adenylate cyclase activating peptide-38) (Fig. 4C), presumably acting through the PAC1 receptor that is expressed on these cells (28). PAC1 receptor is a G proteincoupled receptor linked to the production of camp and activation of PKA. In these cells PACAP ability to inhibit ERKs required PKA, as it was blocked by the expression of the PKA JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 NUMBER 38 SEPTEMBER 20, 2013

6 FIGURE 4. camp inhibits ERK activation and cellular proliferation in H1299 and HCT116 cells. A, camp inhibits the basal ERK activation in H1299. H1299 cells were serum-starved overnight and treated with F/I for the indicated times. MEK activation was measured using phosphorylation-specific antibodies against activated MEK (pmek)(first panel). ERK activation was detected by Western blot using phosphorylation-specific antibodies (perk)(second panel). Total levels of ERK2 are shown in the third panel. B, camp inhibits growth factor stimulated ERK activation in H1299 cells. Cells were serum-starved overnight and treated with EGF (1 ng/ml) for the indicated times in the presence and absence of 10 min F/I pretreatment. ERK activation (perk) was detected by Western blot using phosphorylation-specific antibodies (perk). perk levels are shown in the top panel. Total levels of ERK2 are shown in the bottom panel. C, PACAP38 inhibits the basal ERK activation in H1299 in a PKA-dependent manner. H1299 cells were transfected with Myc-ERK2 in the presence or absence of protein kinase inhibitor (PKI), a selective peptide inhibitor of PKA. After serum starvation, cells were treated with F/I or PACAP38. Myc-ERK2 activation (pmyc-erk2) was measured within the Myc IP by Western blot using phosphorylation-specific antibodies. D, camp inhibits the proliferation of H1299 cells. H1299 cells were plated, serum-starved, and treated with F/I (F) and/or UO126 (UO) for 3 days, and cell numbers were assessed by MTT assay. Relative cell numbers after 3 days are shown as the percent of cell number compared with the untreated group. Normalized averages of the three different experiments are shown S.E. There was statistical significance between all conditions (p 0.05) except those marked with ns (not significant). E, camp inhibits the basal ERK activation in HCT116 cells. Cells were serum-starved overnight and treated with F/I for the indicated times. MEK activation was measured using phosphorylation-specific antibodies against activated MEK (pmek)(first panel). ERK activation was detected by Western blot using phosphorylation-specific antibodies (perk)(second panel). Total levels of ERK2 are shown in the third panel. F, camp inhibits cellular proliferation in HCT116 cells. HCT116 cells were plated, serum-starved, and treated with F/I (F), UO126 (UO), or both (F/UO) for 3 days, and cell numbers were assessed by MTT assay. Relative cell numbers after 3 days are shown as the percent of cell number compared with the untreated group. Normalized averages of the three different experiments are shown S.E. There was statistical significance between all conditions (p 0.05). inhibitor protein kinase inhibitor (Fig. 4C). In H1299 cells, treatment with F/I also blocked proliferation (Fig. 4D). This block was similar to that seen using the MEK inhibitor UO126. Moreover, F/I had a very small additive effect when combined UO126 (Fig. 4D), suggesting that F/I was functioning largely by blocking ERK signaling. Similar inhibitions of MEK and ERK activity (Fig. 4E) and proliferation (Fig. 4F) by F/I were seen in HCT116 cells. The inhibition of ERKs by camp was also seen in non-cancerous and primary cells, including T lymphocytes, Beas-2b lung epithelial cells, and NIH3T3 cells. In these experiments, ERK activity was triggered by the addition of exogenous stimulators of Ras-dependent signaling (Fig. 5, A, B, and C). PKA Inhibits Ras Binding to Both C-Raf and B-Raf in H1299 Cells PKA inhibition of ERKs is thought to be mediated by PKA inhibition of C-Raf binding to Ras (29). However, if B-Raf contributes to ERK activation, any mechanism explaining how PKA inhibits ERKs must include B-Raf as well. Initially, we examined whether B-Raf binding to Ras was a target of inhibition by camp/pka in Ras-mutant cells. In H1299 cells, endogenous Ras proteins bound constitutively to B-Raf (Fig. 6A) as well as to C-Raf (Fig. 6B), and both interactions were blocked by treatment with F/I (Fig. 6, A and B). Similar results were seen using transfected NRasV12 in both H1299 and HCT116 cells (Fig. 6, C and D). We also examined Hek293 cells stimulated by growth factors. In Hek293 cells, activation of wild type NRas by EGF induced B-Raf binding, and this was blocked by F/I (Fig. 6E). Therefore, the inhibition of B-Raf binding to Ras by camp was also seen during activation of wild type Ras. B-Raf binding to the small G protein Rap1 has been shown to occur during camp-dependent activation of ERKs (30, 31). Therefore, we also determined whether B-Raf binding to activated Rap1 was inhibited by camp. For these experiments we used RapE63, a constitutively active mutant of Rap1. Unlike that seen for B-Raf binding to RasV12, forskolin treatment had no significant effect on the ability of B-Raf to bind RapE63 (Fig. 7, A C). This suggests that RapE63, but not Ras, can couple to B-Raf during PKA stimulation of ERKs. SEPTEMBER 20, 2013 VOLUME 288 NUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 27651

7 FIGURE 5. camp inhibits ERK activation in multiple non-cancerous cell types. A, camp inhibits the stimulated ERK activation in primary T cells. CD4-positive T cells were harvested from mouse spleen. Cells were plated and stimulated with either anti-cd3 cross-linking or phorbol-12-myristate-13-acetate in the presence and absence of 10 min of F/I pretreatment. ERK activation was detected by Western blot using phosphorylation-specific antibodies (perk). Total levels of ERK2 are shown as controls. B, camp inhibits both the basal and stimulated ERK activation in Beas-2b lung epithelial cells. Cells were plated and stimulated with EGF for 20 min in the presence and absence of 10 min F/I pretreatment. ERK activation was detected by Western blot using phosphorylation-specific antibodies (perk). Total levels of ERK2 are shown as controls. C, camp inhibits the stimulated ERK activation in NIH3T3 cells. Cells were plated and stimulated with PDGF for 5 min in the presence and absence of 10 min F/I pretreatment. MEK activation was measured using phosphorylation-specific antibodies against activated MEK (pmek)(first panel). ERK activation was detected by Western blot using phosphorylation-specific antibodies (perk)(second panel). Total levels of ERK2 are shown in the third panel. FIGURE 6. camp inhibits the binding between B-Raf and Ras in H1299 and HCT116 cells. A, camp inhibits the binding between endogenous B-Raf and NRas in H1299 cells. H1299 cells were treated with F/I or left untreated as indicated. Cell lysates were subjected to IP with a control IgG (first lane) or a pan-ras antibody (second and third lanes). The levels of endogenous B-Raf within each IP are shown in the first panel, and the levels of endogenous NRas within each IP are shown in the second panel. The input levels of B-Raf and NRas are shown in the third and fourth panels, respectively. B, camp inhibits the binding between endogenous C-Raf and NRas in H1299 cells. H1299 cells were treated with F/I or left untreated as indicated. Cell lysates were subjected to IP with a control IgG (first panel) or an NRas-specific antibody (second and third panels). The levels of endogenous C-Raf within each IP are shown in the first panel, and the levels of endogenous NRas within each IP are shown in the second panel. The input levels of C-Raf and NRas are shown in the third and fourth panels, respectively. C, camp inhibits the binding of both endogenous B-Raf and C-Raf to FLAG-NRasV12 in H1299 cells. H1299 cells were transfected with empty vector (first panel) or FLAG-NRasV12 (second through fourth lanes). After serum starvation, cells were treated with F/I as indicated, and lysates were subjected to FLAG IP. The levels of B-Raf and C-Raf within the IPs are shown in the first and second panels, respectively, and the levels of FLAG-NRasV12 within each IP are shown in the third panel. The fourth and fifth panels show the input levels of endogenous B-Raf and C-Raf, respectively. The sixth panel shows the input level of transfected FLAG-NRasV12. D, camp inhibits the binding of both endogenous B-Raf and C-Raf to FLAG-NRasV12 in HCT116 cells. HCT116 cells were transfected with empty vector (first panel) or FLAG-NRasV12 (second through fourth lanes). After serum starvation, cells were treated with F/I as indicated, and lysates were subjected to FLAG IP. The levels of B-Raf and C-Raf within the IPs are shown in the first and second panels, respectively, and the levels of FLAG-NRasV12 within each IP are shown in the third panel. The fourth and fifth panels show the input levels of endogenous B-Raf and C-Raf, respectively. The sixth panel shows the input level of transfected FLAG-NRasV12. E, camp blocks EGF stimulation of B-Raf binding to wild type FLAG-NRas (WT) in Hek293 cells. All cells were transfected with FLAG-NRas WT. After serum starvation, cells were treated with F/I or EGF as indicated and subjected to FLAG IP. The level of endogenous B-Raf within the IP is shown in the first panel, and the levels of WT FLAG-NRas within each IP are shown in the second panel. The input levels of endogenous B-Raf and transfected WT FLAG-NRas are shown in the third and fourth panels, respectively. PKA Phosphorylation of Serine 259 within C-Raf Prevents Binding to Ras As mentioned above, C-Raf is a well-established target of PKA inhibition. A number of phosphorylation sites in C-Raf have been suggested to contribute to PKA ability to inhibit Ras signaling to C-Raf, including phosphorylations of serine 259 (Ser(P)-259) and serine 43 (Ser(P)-43) in C-Raf (32 34). Phosphorylation of Ser-259 serves as an amino-terminal binding site for the adaptor protein (35). Phosphorylation of Ser-43 of C-Raf by PKA may interfere with C-Raf binding to Ras (36), but others studies have challenged this view (32, 34, 37). Here, we confirmed the role of Ser(P)-259 in PKA inhibition of C-Raf binding to Ras in H1299 cells. We show the ability of F/I to inhibit NRas binding to C-Raf was greatly reduced in the C-Raf mutant lacking this phosphorylation site (C-Raf S259A) (Fig. 8A). In contrast, F/I treatment was able to inhibit binding of Ras to C-Raf S43A mutant to the same level as that shown for wild type C-Raf (Fig. 8A), confirming that phosphorylation of JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 NUMBER 38 SEPTEMBER 20, 2013

8 FIGURE 7. camp does not inhibit the binding between B-Raf and Rap1. A, camp inhibits the binding of B-Raf to NRasV12 in Hek293 cells. Hek293 cells were transfected with FLAG-NRasV12 and GFP-B-Raf. After serum starvation, cells were treated with F/I as indicated and subjected to FLAG IP. The levels of GFP-B-Raf within each IP are shown in the first panel, and the levels of FLAG-NRasV12 within each IP are shown in the second panel. The input levels of GFP-B-Raf (GFP) and transfected FLAG-NRasV12 (FLAG) are shown in the third and fourth panels, respectively. B, camp does not inhibit the binding of B-Raf to RapE63 in Hek293 cells. Hek293 cells were transfected with FLAG-RapE63 and GFP-B-Raf. After serum starvation, cells were treated with F/I as indicated and subjected to FLAG IP. The levels of GFP-B-Raf within each IP are shown in the first panel, and the levels of FLAG-RapE63 within each IP are shown in the second panel. The input levels of GFP-B-Raf (GFP) and transfected FLAG-RapE63 (FLAG) are shown in the third and fourth panels, respectively. C, the results of A and B are presented as the percent of B-Raf associating with NRasV12 or RapE63 treated with F/I ( ) or left untreated ( ). Untreated ( ) is 100%. Quantitation of binding was performed by ImageJ and normalized to total FLAG protein within the IP. The normalized data are represented as the mean S.E from three independent experiments. ns, not significant. *, statistical significance is Ser-259 plays a dominant role in PKA inhibition of C-Raf binding to Ras in these cells. These results are in general agreement with previous studies that phosphorylation of Ser-259 creates a binding site for the adaptor protein , and binding at that site blocks Ras binding (38 40). PKA Phosphorylation of Ser-365 within B-Raf Prevents B-Raf Binding to Ras The mechanism by which PKA inhibits B-Raf binding to Ras is not known. We show that PKA inhibition of B-Raf binding to Ras requires phosphorylation of B-Raf on serine 365. B-Raf Ser-365 is analogous to the Ser-259 site in C-Raf that is required for PKA inhibition (32, 39). B-Raf S365A, a mutant B-Raf lacking this phosphorylation site, bound to NRasV12 in H1299 cells, but this binding was not inhibited by F/I (Fig. 8B). F/I blocked the activation of ERKs in cells transfected with B-Raf WT but not B-Raf S365A (Fig. 8C). These data suggest that PKA phosphorylation of Ser-365 in B-Raf uncouples NRas from B-Raf and prevents B-Raf from activating ERKs. The phospho-specific antibody designed against Ser(P)-259 in C-Raf can also be used to monitor Ser(P)-365 in B-Raf (41). Using this antibody, Ser-365 phosphorylation was detected basally, and the level of this phosphorylation was increased upon forskolin treatment (Fig. 8D). Phosphorylation at Ser-365 has been shown to induce the binding of B-Raf to (35), and F/I also increased the level of bound to B-Raf (Fig. 8E). Moreover, the absence of phosphorylation at this site in the mutant B-Raf S365A greatly reduced binding and prevented the increase in binding induced by forskolin (Fig. 8E). We suggest that the basal binding of B-Raf to Ras occurs within the pool of B-Raf that is basally unphosphorylated at Ser-365. We propose that the PKA-dependent increase in the phosphorylation on Ser-365 upon activation of PKA and the resultant increase in binding to this phosphorylated site prevents B-Raf from binding to oncogenic Ras. This is similar to the model proposed for the regulation of Ras/C-Raf association via binding of to phosphorylated Ser-259 within C-Raf (39). DISCUSSION Ras-mutant cancers and cell lines maintain their dependence on Ras signaling in a process that is referred to as oncogene addiction (25, 42, 43). Signaling downstream of oncogenic Ras along the MAP kinase cascade can be mediated by either C-Raf and/or B-Raf. We show here that both B-Raf and C-Raf participate in ERK activation in multiple Ras-mutant cancer cell lines. This finding is different from that reported in recent studies showing that B-Raf does not participate in ERK activation in Ras-mutant melanomas (9). The finding that B-Raf can still bind Ras and contribute to ERK activation in some non-melanocytic cancer cells is relevant because it demonstrates that cancers cells harboring Ras mutations are not always dependent on C-Raf signaling. This knowledge is important because of the growing potential of B-Raf-specific therapies. Studies in melanomas have received much recent attention for the role of Raf dimerization in ERK activation by oncogenic Ras and specific Raf inhibitors (6, 44, 45). Although it was not the focus of this study, we show that the binding of B-Raf to Ras was independent of B-Raf dimerization, as similar binding to Ras was seen in wild type B-Raf and dimerization-deficient mutants of B-Raf. The uncoupling of B-Raf from Ras in Ras-mutant melanoma cells is due to the phosphorylation of B-Raf on a specific ERK target site (Ser-151) that lies near the Ras-binding site of B-Raf (10). This site is basally phosphorylated within B-Raf in Rasmutant melanoma cells, and this phosphorylation is thought to prevent it from binding to oncogenic Ras (10). Here, we confirm those results and the results of others (26) that phosphorylation of Ser-151 largely excludes B-Raf from associating with Ras. However, in H1299 cells the basal levels of Ser(P)-151 were quite low. This low level of phosphorylation presumably accounts for the high level of basal association of B-Raf with active Ras. In non-melanoma Ras-mutant cells, we show findings that B-Raf directly associates with activated Ras and that Ser-151 is not highly phosphorylated. Prior studies in melanoma cells have shown that the phosphorylation of Ser-151 is extremely stable due to its slow rate of dephosphorylation (10). This may be because the dephosphorylation of Ser(P)-151 requires both the phosphatase PP2A and the proline isomerase Pin1 (26). Pin1 expression is elevated in many human cancers (46), including NSCLCs (47, 48), and can enhance the transforming SEPTEMBER 20, 2013 VOLUME 288 NUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 27653

9 FIGURE 8. The ability of camp to block Raf association with Ras is regulated by phosphorylation of serine 259 in C-Raf and serine 365 in B-Raf. A, camp decreases the interaction between NRasV12 and C-Raf WT but not the interaction between NRasV12 and C-Raf S259A. H1299 cells were transfected with FLAG-NRasV12 and Myc-C-Raf (first and second lanes), Myc-C-Raf S43A (third and fourth lanes), or Myc-C-Raf S259A (fifth and sixth lanes). After serum starvation, the cells were treated with F/I or left untreated as indicated, and lysates were subjected to FLAG IP. The presence of Myc-C-Raf within each IP is shown in the first panel. The presence of FLAG-NRasV12 within the FLAG IP is shown in the second panel. The third and fourth panels show the input levels of Myc-C-Raf (WT or mut) and FLAG-NRasV12, respectively. B, camp decreases the interaction between FLAG-NRasV12 and HA-B-Raf wild type but not HA-B-Raf S365A. H1299 cells were transfected with FLAG-NRasV12 with either wild type (WT) HA-B-Raf (first and second lanes) or with HA-B-Raf S365A (third and fourth lanes). After serum starvation, the cells were treated with F/I or left untreated as indicated, and lysates were subjected to FLAG IP. The presence of HA-B-Raf within each IP is shown in the first panel. The levels of FLAG-NRasV12 with each IP are shown in the second panel. The third and fourth panels show the input levels of HA-B-Raf (WT or mut) and FLAG-NRasV12, respectively. C, camp decreases the activation of ERK induced by HA-B-Raf wild type but not S365A mutant. H1299 cells were transfected with vector, HA-B-Raf, or HA-B-Raf S365A along with FLAG-ERK2. After serum starvation, cells were treated with F/I or left untreated as indicated, and lysates were subjected to FLAG IP. FLAG-ERK2 activation within the IP was measured by Western blot using perk antibodies (top panel). The levels of FLAG-ERK2 within each IP are shown in the second panel. The input levels of transfected HA-B-Raf (WT or mut) and FLAG-ERK2 are shown in the third and fourth panels, respectively. D, B-Raf was basally phosphorylated on Ser-365 and further phosphorylated on Ser-365 after camp treatment. H1299 cells were transfected with FLAG-B-Raf wild type. After serum starvation, cells were treated with F/I for the indicated times, and lysates were subjected to FLAG IP. The phosphorylation of Ser-365 (pser365) was detected in the IP using a phospho-specific Ab recognizing B-Raf Ser(P)-365 (first panel). The levels of FLAG-B-Raf within each IP are shown in the second panel. The levels of FLAG-B-Raf within the total lysates (Input) are shown in the third panel. E, camp increases the interaction between FLAG-B-Raf and Myc but not the interaction between FLAG-B-Raf (S365A) and Myc H1299 cells were transfected with Myc with either wild type FLAG-B-Raf (first and second lanes) or FLAG-B-Raf S365A (third and fourth lanes). After serum starvation, cells were treated with F/I or left untreated as indicated, and lysates were subjected to FLAG IP. The levels of Myc within the IP are shown in the first panel, and the levels of FLAG-B-Raf (WT or mut) within the IP are shown in the second panel. The third and fourth panels show the input levels of Myc and FLAG-B-Raf (WT or mut), respectively. ability of Ras activation (49, 50). The levels of Pin1 activity may help determine the steady state levels of phosphorylation of Ser-151 seen in different Ras-mutant tumors, influencing the contribution of B-Raf to Ras-dependent signaling. The reliance of Ras-transformed melanoma cells on C-Raf has been proposed to make the melanoma cells particularly vulnerable to camp/pka inhibition of ERKs and cell proliferation (9, 10). This model suggests that B-Raf activation via Ras is immune to the inhibitory actions of PKA that characterize C-Raf. The finding that B-Raf can bind Ras to participate in Ras activation of ERKs and proliferation in some Ras-mutant tumor cells permits us to ask a longstanding question. Is PKA inhibition of Ras signaling specific for C-Raf? Or is B-Raf equally vulnerable? Indeed, despite reports to the contrary (51), it has long been assumed that, among C-Raf and B-Raf isoforms, only Ras binding to C-Raf is inhibited by PKA (29, 52). This view has arisen in parallel with the parallel view that only B-Raf is capable of mediating a signal from camp/pka to JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 NUMBER 38 SEPTEMBER 20, 2013

10 ERKs. Indeed, in many cells types B-Raf is required for ERK activation through hormones coupled to camp/pka, including melanocytes (53), renal collecting duct cells (54), prostate cells (55), chondrocytes (56), thyroid cells (57), renal cells (58), pituitary cells (59), and neuronal cells (31, 60, 61). There are cases where B-Raf does not seem to be involved, however (62, 63). One PKA phosphorylation site that has been proposed to mediate the selective inhibition of C-Raf by PKA is Ser-43 (33, 36). When this site is phosphorylated, this site blocks access to Ras (36). Because the analogous site in B-Raf (Ser-151) is not a PKA target, this finding helped perpetuate the belief that Ras binding to C-Raf and C-Raf is differentially regulated by PKA. We show here that Ser-43 phosphorylation is not required for PKA ability to inhibit C-Raf binding to Ras. In contrast, the phosphorylation of Ser-259 is required for this inhibition of C-Raf binding to Ras by PKA. This finding is in general agreement with Dumaz and Marais (40). We also show that the mechanism for this uncoupling of B-Raf from Ras is the same as that proposed for C-Raf. The site in B-Raf (Ser-365) that is analogous to Ser-259 is also a target for PKA phosphorylation, and this phosphorylation is required for PKA inhibition of B-Raf binding to Ras as well. Although the similar consequences of PKA phosphorylation of these two sites may not be surprising given the sequence homologies between the two isoforms, it provides formal proof that PKA inhibits the interactions of both C-Raf and B-Raf with Ras. Why does camp inhibit ERK signaling in some cells but activates ERKs in other cells? Given that PKA can inhibit both C-Raf and B-Raf effector pathways coupling Ras to ERKs, it is clear that camp/pka activation of ERKs must utilize a different pathway. Indeed, it has been proposed that the ability of camp/pka to activate or inhibit ERKs in B-Raf-expressing cells is dependent on additional factors. For example, the level of proteins associating with B-Raf has been proposed to dictate whether PKA can activate or inhibit ERKs (64). Other models predict that the levels of the scaffold molecules including KSR (65) and the PKA anchoring protein AKAP-Lbc (66) will influence how PKA regulates ERKs. Ras-independent mechanisms of camp/pka activation of ERKs have also been proposed. In many B-Raf-expressing cells, camp/pka activation of ERKs is mediated by the small G protein Rap1 (23, 30, 31, 54 56, 59, 67 69). We show that PKA does not disrupt the association between Rap1 and B-Raf. Therefore, the mechanism of Ras-dependent inhibition of ERKs by PKA described here may not occur during Rap1 signaling to ERKs. The presence of oncogenic Ras mutations renders cancer cells dependent on Ras signaling (25). Therefore, PKA-dependent inhibition of Ras signaling to both B-Raf and C-Raf will likely dominate PKA actions on the Ras-dependent basal ERK activity in these cells. This is similar to the inhibitory effect of PKA on growth factor activation of ERKs that has been seen in a number of cell lines (70). In these examples, PKA ability to stimulate ERKs in the absence of added growth factors is masked by the much larger effect of PKA to inhibit ERK activation in the presence of added growth factors. In summary, we show that oncogenic mutant forms of Ras in human cancers may still bind to B-Raf. This explains why these tumors maintain their dependence on both C-Raf and B-Raf. It also identifies a potential target of PKA inhibition of ERK, the interaction between Ras and B-Raf. Identifying the mechanism by which PKA can disrupt B-Raf binding to Ras has implications for treatment of cancers harboring oncogenic Ras mutations. These studies suggest that camp and hormones coupled to camp might be effective in dampening the effects of oncogenic Ras in non-melanocytic cancer cells, even in cases where Ras/B-Raf signaling participates in the basal proliferation of these cells. Importantly, mechanisms that dismantle endogenous camp signaling would be expected to provide a growth advantage in these cancers, as has been shown in some melanomas (10). Acknowledgments We acknowledge Zhiping Wang and Tara J. Dillon for critically reading the manuscript. REFERENCES 1. Prior, I. A., Lewis, P. D., and Mattos, C. (2012) A comprehensive survey of Ras mutations in cancer. Cancer Res. 72, Bos, J. L. (1989) ras oncogenes in human cancer. A review. Cancer Res. 49, Ji, Z., Flaherty, K. T., and Tsao, H. (2012) Targeting the RAS pathway in melanoma. Trends Mol. Med. 18, Rinehart, J., Adjei, A. A., Lorusso, P. M., Waterhouse, D., Hecht, J. R., Natale, R. B., Hamid, O., Varterasian, M., Asbury, P., Kaldjian, E. 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Cancer Res. 62, Forbes, S. A., Bindal, N., Bamford, S., Cole, C., Kok, C. Y., Beare, D., Jia, M., Shepherd, R., Leung, K., Menzies, A., Teague, J. W., Campbell, P. J., Stratton, M. R., and Futreal, P. A. (2011) COSMIC. Mining complete cancer genomes in the catalogue of somatic mutations in cancer. Nucleic Acids Res. 39, D945 D950 SEPTEMBER 20, 2013 VOLUME 288 NUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 27655