Mutations of C-RAF Are Rare in Human Cancer because C-RAF Has a Low Basal Kinase Activity Compared with B-RAF

The protein kinase B-RAF is mutated in f8% of human cancers. Here we show that presumptive mutants of the closely related kinase, C-RAF, were detected in only 4 of 545 (0.7%) cancer cell lines. The activity of two of the mutated proteins is not significantly different from that of wild-type C-RAF and these variants may represent rare human polymorphisms. The basal and B-RAF–stimulated kinase activities of a third variant are unaltered but its activation by RAS is significantly reduced, suggesting that it may act in a dominant-negative manner to modulate pathway signaling. The fourth variant has elevated basal kinase activity and is hypersensitive to activation by RAS but does not transform mammalian cells. Furthermore, when we introduce the equivalent of the most common cancer mutation in B-RAF (V600E) into C-RAF, it only has a weak effect on kinase activity and does not convert C-RAF into an oncogene. This lack of activation occurs because C-RAF lacks a constitutive charge within a motif in the kinase domain called the N-region. This fundamental difference in RAF isoform regulation explains why B-RAF is frequently mutated in cancer whereas C-RAF mutations are rare. (Cancer Res 2005; 65(21): 9719-26)


Introduction
The protein kinases of the RAF family, mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) family, and ERK family form a three-tiered cascade that is activated in a RAS-dependent manner and which is an important regulator of cell fate decisions (1,2).There are three RAF proteins in mammals, A-RAF, B-RAF, and C-RAF, and they share three conserved regions: CR1 and CR2 within the regulatory NH 2 terminus and CR3 encompassing the kinase domain within the COOH terminus (see Fig. 1).RAF proteins are normally cytosolic but they are recruited to the plasma membrane by the small G-protein RAS, and this is an essential step for their activation by growth factors, cytokines, and hormones.At the membrane, RAF activation occurs through a highly complex process involving conformation changes, binding to other proteins, binding to lipids, and phosphorylation and dephosphorylation of some residues (3,4).
Within the kinase domain, phosphorylation of two motifs is required for activation.One of these is called the activation segment, which must be phosphorylated on conserved threonine and serine residues.In B-RAF, these are T599 and S602 (originally designated as T598 and S601 due to a sequencing error in the database; ref. 5) and in C-RAF, the corresponding residues are T491 and S494.Structural studies have revealed that when the activation segment is not phosphorylated, it binds to another region of the kinase domain called the glycine-rich loop, trapping B-RAF in an inactive conformation.T599 phosphorylation is thought to disrupt this interaction, releasing the activation segment and reorientating critical residues into the correct position for catalysis (6).Presumably, similar mechanisms operate in the other isoforms.
The other motif that must be phosphorylated is called the negative-charge regulatory or N-region.The N-region controls a fundamental difference in how the RAF proteins are regulated.In C-RAF, the N-region sequence is 338 SSY 341 Y and phosphorylation of S338 and Y341 is essential for activation by RAS and growth factors.Both sites are conserved in A-RAF (S299 and T302, respectively) but in B-RAF, Y340 and Y341 are replaced by aspartic acids (D448 and D449) and although S338 is conserved, it is constitutively phosphorylated (7).All RAF isoforms are only fully activated when four negative charges occupy the N-region, either from the phosphorylated serine and tyrosine or from the phosphorylated serine and aspartic acids.However, whereas the charges are present constitutively in B-RAF, in A-RAF and C-RAF they are only acquired under activating conditions.Consequently, the basal kinase activity of B-RAF is considerably higher than that of A-RAF and C-RAF and whereas the latter two need both RAS and SRC for activation, B-RAF is fully activated by RAS alone (8).Importantly, for all isoforms, activation segment phosphorylation and, in the case of A-RAF and C-RAF, N-region phosphorylation occur at the plasma membrane, in part explaining why membrane recruitment is essential for RAF activation by RAS and membrane-bound receptors.
We recently described a systematic sequencing approach that allowed us to identify somatic gain-of-function mutations in the B-RAF gene in f8% of human cancers (5,9).Importantly, a glutamic acid substitution for the valine at codon 600 (V600) accounts for f90% of the B-RAF mutations, demonstrating extraordinary selection for this genetic lesion.V600E B-RAF is activated f500-fold; it stimulates constitutive MEK-ERK signaling in cells and transforms fibroblasts and melanocytes (6,(9)(10)(11)(12).Over 45 other cancer-associated mutations have been described in B-RAF, the majority clustering to the glycine-rich loop and the activation segment, the two regions of the kinase domain that are responsible for trapping B-RAF in the inactive conformation (5).
These mutations are thought to activate B-RAF by disrupting the inactive conformation of the kinase and allowing the active conformation to prevail (6).
It is clear that B-RAF is important in human cancer but the role(s) of A-RAF and C-RAF is less evident.In this study, we screen 545 cancer cell lines and over 100 tumor samples for mutations in A-RAF and C-RAF.We describe four coding region changes that were identified in C-RAF but do not find any such mutations in A-RAF.However, these presumed mutations in C-RAF have weak effects on C-RAF kinase activity and none of them converts C-RAF into a transforming oncogene.We have also created a V492E substitution in C-RAF to mimic the common V600E mutation of B-RAF and find that this also has only weak kinase activity and lacks NIH 3T3 transforming activity.The lack of C-RAF activation by these mutations seems to be due to the lack of intrinsic charge within the N-region, demonstrating that this motif reveals the oncogenic potential of B-RAF and explaining why C-RAF is not frequently mutated in human cancer.

Materials and Methods
Genomic sequencing was done as described (9) using the primers shown in Supplementary Table 1.Expression constructs for G12 VRAS, C-RAF, and B-RAF have been described (8).Additional B-RAF and C-RAF substitutions were generated by PCR mutagenesis and verified using automated dideoxy sequencing.COS7 cells and NIH 3T3 cells were maintained in DMEM (Life Technologies, Paisley, Scotland) supplemented with 10% or 5% FCS, respectively.COS7 cells were transfected with LipofectAMINE (Invitrogen, Carlsbad, CA) as described (8).NIH 3T3 transformation assays were done as described (6).Preparation of COS cell lysates, Western blotting, protein expression measurements, and RAF-coupled kinase assay have all been described previously (7,8,13).Blotting for pS338 phosphorylated C-RAF was done as described (7).Blotting for phosphorylated MEK was done using standard techniques and a ppMEK1/2 antibody (9121L, Cell Signaling Technology, Beverly, MA).

Results
C-RAF mutations are rare in human cancer.We previously identified 43 mutations in the B-RAF gene when we sequenced the exons and intron-exon boundaries in 545 cancer cell lines but did not observe any mutations in 341 normal DNA samples (9).Here we have also sequenced all 16 exons of the C-RAF gene and all 15 exons of the A-RAF gene in these cancer cell lines and normal DNA samples, comparing the data to the sequences of A-RAF and C-RAF available on the National Center for Biotechnology Information database. 3Whereas 7.9% of the cancer cell lines have mutations in B-RAF, only four (0.73%) have coding region variations that result in amino acid changes in C-RAF, and there were no such mutations in A-RAF.We did not observe any such changes in either A-RAF or C-RAF in the normal DNA samples.The presumptive C-RAF mutations are a serine for proline substitution at position 207 (P207S) in SW684 fibrosarcoma cells; an isoleucine for valine at 226 (V226I) in ChaGo-K-1 lung carcinoma cells; a histidine for glutamine at 335 (Q335H) in NCI-H2087 lung adenocarcinoma cells; and a lysine for glutamic acid at 478 (E478K) in Ls513 colorectal carcinoma cells (Table 1).
We have also sequenced A-RAF and C-RAF from primary tumors from 18 breast cancers, 26 lung cancers, 15 testis cancers, and 20 gastric cancers.In addition, we have sequenced C-RAF from 30 colorectal cancers and its kinase domain from an additional 10 gliomas, 9 acute lymphoid leukemias, and 20 myeloproliferative disease samples.Finally, we have sequenced the A-RAF kinase domain from 10 gliomas.We have not detected either A-RAF or C-RAF mutations in any of these primary tumors, again demonstrating that C-RAF mutations are rare and A-RAF mutations are rare or nonexistent.
The P207S, V226I, and E478K substitutions do not occur coincident with mutations in either B-RAF or any of the RAS genes whereas Q335H occurs coincident with activating mutations in both B-RAF (L597V) and N-RAS (Q61K) in NCI-H2087 cells (Table 1).P207 and V226 are between CR1 and CR2 in a region of the protein of which function is unknown; Q335 is between CR2 and CR3, three residues upstream of the N-region; and E478K is within the kinase domain (Fig. 1).P207, V226, and Q335 are reasonably well, but not absolutely, conserved in C-RAF from several species but they are not conserved in A-RAF or B-RAF from any species or, with the exception of Q335 in Caenorhabditis elegans, in the single RAF paralogues from lower organisms (Fig. 1B).In contrast, E478 is highly conserved, with glutamic acid being found at the corresponding position in all RAF orthologues and paralogues from lower and higher organisms.Indeed, a review of the human genome reveals that 27.6% of all human kinases possess a glutamic or aspartic acid at the corresponding position (data not shown), demonstrating a strong selection for an acidic amino acid at this position in many kinases.Importantly, there are no reports showing that the amino acids in B-RAF that correspond to P207, V225, and Q335 (T312, T330, and R443, respectively) are mutated in human cancer whereas the equivalent of E478 (E586 in B-RAF) is mutated and, intriguingly, also to a lysine (9).E586K B-RAF has f130-fold elevated kinase activity (6).
P207S, V226I, and Q335H substitutions do not activate C-RAF.For characterization, we expressed myc-epitope tagged versions of these presumptive C-RAF mutants in COS cells and measured their activity in a kinase cascade assay using glutathione S-transferase (GST)-MEK, GST-ERK, and myelin basic protein as substrates, with ATP at a physiologically relevant concentration of 5 mmol/L (6).Under these conditions, C-RAF has low basal kinase activity but is activated 80-to 120-fold by oncogenic RAS ( G12V RAS; Fig. 2A and B).The basal kinase activities of P207S C-RAF and V266I C-RAF are similar to that of WT C-RAF and their activation by G12V RAS is also similar to that of WT C-RAF although it was consistently lower (Fig. 2A and B).In line with their lack of elevated basal kinase activity, these mutants do not activate MEK in COS cells (Fig. 2C) and they do not transform NIH 3T3 cells (Fig. 2D).
The basal kinase activity of Q335H C-RAF is also not elevated compared with that of WT C-RAF (Fig. 2A) and this variant also fails to activate MEK in COS cells (Fig. 2C) or to transform NIH 3T3 cells (Fig. 2D).However, Q335H C-RAF activation by G12V RAS was impaired and only reached 20% to 25% of the levels seen with WT C-RAF (Fig. 2B).Because Q335 is only three amino acids upstream of the N-region of C-RAF (Fig. 1A), we examined if the reduced activation of Q335H C-RAF is due to disrupted S338 phosphorylation.Using an antibody that only binds to C-RAF when S338 is phosphorylated (7), we find that antibody binding is significantly reduced in Q335H C-RAF (Fig. 3A).One interpretation of this result is that the Q335H substitution disrupts antibody binding and not S338 phosphorylation; thus, we used an alaninescan mutagenic approach to define that epitope.Antibody binding is disrupted when R336, D337, and S338 are mutated to alanine, but importantly not when Q335 is mutated (Fig. 3B).Thus, the antibody epitope seems to be ''RDpS'' and we conclude that Q335 is not part of it and that S338 phosphorylation on Q335H C-RAF is reduced in the presence of G12V RAS.Finally, because Q335H C-RAF occurs coincident with L597V B-RAF in NCI-H2087 cells (Table 1) and we recently reported the surprising finding that mutant forms of B-RAF can activate WT C-RAF (6), we tested whether Q335H C-RAF can be activated by L597V B-RAF.Unlike the differences observed with G12V RAS, L597V B-RAF activates Q335H C-RAF and WT C-RAF to similar levels (Fig. 3C).
The E478K substitution activates C-RAF.In contrast to the other presumptive mutants, the E478K variant does have elevated kinase activity, its basal kinase activity being 25-fold higher than that of WT C-RAF (Fig. 4A).The activation of C-RAF by E478K compares poorly to the enhanced activity of E586K B-RAF, which we have shown is f130-fold more active than WT B-RAF (6).Importantly, whereas E586K B-RAF stimulates strong constitutive MEK-ERK signaling in COS cells (6,14), MEK activation by E478K C-RAF is weak (Fig. 4B); furthermore, whereas E586K B-RAF transforms NIH 3T3 cells, E478K C-RAF does not (Fig. 4C).Remarkably, however, whereas WT C-RAF is activated f100-fold by G12V RAS, E478K C-RAF is activated over 500-fold (Fig. 4A), producing a level of activation that is an impressive 13,000-to 14,000-fold higher than the basal activity of WT C-RAF.Thus, E478K C-RAF is hypersensitive to activation by oncogenic RAS, suggesting that this mutant can be strongly activated by secondary signals.
We were intrigued by the distinct responses of C-RAF and B-RAF to the E478K/E586K mutations and wished to investigate the molecular mechanism underlying this difference.As described in Introduction, a key difference in regulation of the RAF proteins is mediated by their N-regions; thus, we tested whether this region determines how C-RAF and B-RAF respond to the E478K/E586K mutations.Y340 and Y341 of C-RAF were substituted with aspartic acids (creating DD C-RAF) to mimic the N-region of B-RAF (Fig. 1B).
We also did the reverse experiment, neutralizing the charge within the N-region of E586K B-RAF by substituting S446, S447, D448, and D449 with alanines ( AAAA,E586K B-RAF).Introducing these substitutions into E586K B-RAF significantly reduces its kinase activity (Fig. 4E) and to dissect the contributions made by the individual charged amino acids within the N-region, we independently substituted S446/S447 and D448/D449 with alanines.Both the S446A/S447A ( AADD,E586K B-RAF) and the D448A/D449A  ( SSAA,E586K B-RAF) substituted proteins have significantly reduced kinase activity (Fig. 4E), demonstrating that S446 phosphorylation and the aspartic acids of the N-region both contribute to the elevated kinase activity of E586K B-RAF.Finally, we show that the transforming activity of E586K B-RAF is significantly reduced when the N-region charge is disrupted (Fig. 4C).We conclude that E478K C-RAF is strongly activated by mutations that introduce a constitutive negative charge into its N-region whereas the activity of E586K B-RAF is compromised by the inverse changes.
C-RAF activation by the V492E substitution is weak because its N-region lacks charge.We were intrigued to note that whereas V600 of B-RAF is mutated in 6% to 7% of human cancers (5), we did not observe any mutations of the equivalent codon (V492) in C-RAF in our 545 cancer cell lines or the 148 primary tumor samples.We therefore tested how a V492E substitution affected C-RAF kinase activity.V492E C-RAF is f45-fold more active than WT C-RAF (Fig. 5A), a level of activation that contrasts strongly with the f500-fold activation seen with the corresponding mutation in B-RAF ( V600E B-RAF; ref. 6).Furthermore, whereas V600E B-RAF stimulates strong constitutive MEK/ERK signaling in mammalian cells (9) and is transforming (Fig. 2D), V492E C-RAF only stimulates weak MEK activity in COS cells (Fig. 5B) and does not transform NIH 3T3 cells (Fig. 5C).
Once again, we were struck by the different responses of C-RAF and B-RAF to this conserved mutation; thus, we examined if this is also due to the lack of N-region charge in C-RAF.When the V492E C-RAF mutation is combined with DD C-RAF ( DD,V492E C-RAF), there is a synergistic augmentation in kinase activity, resulting in a kinase of which basal activity is elevated over 1,000-fold (Fig. 6A).There is also a synergistic activation of MEK in COS cells (Fig. 6B) and a synergistic augmentation of transforming activity (Fig. 6C).
Finally, we did the reverse experiment, neutralizing the Nregion of V600E B-RAF by creating AAAA,V600E B-RAF.This causes an f15-fold reduction in kinase activity but we note that the kinase is still f35-fold more active than WT B-RAF (Fig. 7A).Importantly, AAAA,V600E B-RAF still activates MEK in COS cells (Fig. 6B) and transforms NIH 3T3 cells (Fig. 7C) albeit at reduced levels.

Discussion
We have identified four coding region variants in C-RAF that occur in human cancer cell lines but are not seen in normal DNA samples.These four variants convert P207 to serine, V226 to isoleucine, Q335 to histidine, and E478 to lysine.Dealing with P207S and V226I first, we did not detect any significant changes in either their basal or RAS-stimulated kinase activity when MEK was used as a substrate.It is possible that these substitutions affect other currently unknown and MEK-independent functions of C-RAF; however, P207 and V226 are located in a region of the protein of which function is unknown and they are not even conserved in all C-RAF orthologues or in other RAF paralogues from a variety of higher and lower organisms.Furthermore, the corresponding residues are not mutated in B-RAF in human cancer.One interpretation of our data is that these variants are rare single-nucleotide polymorphisms that play no role in the growth of the cancer cell lines in which they were identified.If they are single-nucleotide polymorphisms, they have not been previously described 4 and we did not observe them in our 341 normal DNA samples, suggesting that they are extremely rare.An alternative explanation is that these are somatic mutations that occur due to collateral DNA damage during the development of these cancers or that have occurred in the cell lines during in vitro culture.Unfortunately, in the absence of control DNA from the patients from whom the cell lines were derived, it is not possible to determine which of these possibilities is correct.Whatever their genesis, it seems that these variants do not have an obvious effect on signaling or a clear effect on proliferation and with the caveat that their effects may only be evident in specific cell types, it seems unlikely that they play a significant role in human cancer.
Q335 is also not conserved in all C-RAF orthologues or in the other RAF paralogues.However, it is three amino acids upstream of the N-region and although this substitution also fails to affect C-RAF basal kinase activity, it does suppress its activation by oncogenic RAS.Our alanine-scan mutagenesis data show that Q335 is not part of the pS338 antibody epitope, which is consistent with the fact that this antibody binds to B-RAF when S446 is phosphorylated (7) and B-RAF has an arginine at the position equivalent to Q335 (Fig. 1B).Our data suggest that the Q335H substitution disrupts S338 phosphorylation in the presence of G12V RAS, causing reduced C-RAF activation.It is not surprising that such a subtle substitution should have such a profound effect.With the exception of Q335, the amino acids surrounding the N-region of C-RAF and B-RAF are well conserved (Fig. 1B), and yet, whereas S338 is only phosphorylated at the plasma membrane, S446 is phosphorylated in the cytosol, demonstrating exquisite selectivity by the kinases involved.Finally, our data show that the Q335H substitution does not affect C-RAF activation by L597V B-RAF, which is consistent with our recent finding that B-RAF activates C-RAF through a distinct mechanism that is largely independent of S338 phosphorylation. 5e have been unable to study the biological function of Q335H C-RAF because we have not been able to apply RNA interference technology to NCI-H2087 cells.However, it has been shown that excessive ERK signaling can induce cell cycle arrest or senescence through induction of cell cycle inhibitory proteins such as p27 and p21 (16)(17)(18)(19)(20)(21).Thus, in cancer, ERK signaling must be kept within narrow limits that are sufficient to stimulate proliferation but not so high as to induce cell cycle arrest.We speculate that in some cancers, mutations or other upstream events that stimulate excessive pathway activity are incompatible with proliferation.This may be a protective mechanism but in these circumstances, precancerous cells must develop strategies to tone down this signaling to progress.We and others recently reported the identification of mutant forms of B-RAF involving residue D594 in human cancer that are devoid of kinase activity (6,12,22).These account for f1% of B-RAF mutations, and thus are too common to be random, and similar mutations are not observed in either C-RAF or A-RAF.Importantly, although coincident mutations in B-RAF and RAS are very rare in human cancer (9), f30% of the inactive mutants occur in cancers that also harbor RAS mutations and we have argued that the D594 mutants could act in a dominantnegative manner to suppress excessive RAS-MEK signaling (5).Thus, it is intriguing that in addition to the Q335H C-RAF substitution, NCI-H2087 cells harbor activating mutations in B-RAF and N-RAS, which is, to our knowledge, the only example of a cancer cell line harboring amino acid changes in three components of this pathway.Perhaps if oncogenic RAS and activated B-RAF both activate C-RAF, MEK signaling is excessive and the Q335H mutation is required to reduce signaling from one of these upstream inputs.Thus, this mutant could be acting in a dominant-negative manner and we are currently developing genetic approaches to test this model.
It is clear that B-RAF is important in human cancer and in the classic sense, it is an oncogene because activated forms can transform immortalized fibroblasts and other cell lines.Critically, ''oncogenic'' B-RAF does not induce cancer by itself because a high proportion of common nevi harbor mutations in the B-RAF gene but are not cancerous (23).Notably, of the variants we identified, only E478K C-RAF has elevated kinase activity, and yet even this mutant does not seem to be a classic oncogene because it does not transform NIH 3T3 cells.However, it is hypersensitive to activation by RAS and synergizes with the Y340D/Y341D double mutations to activate C-RAF and augment transforming activity.Thus, perhaps the oncogenic potential of E478K C-RAF is only apparent in some cell contexts.In common with P207S C-RAF and V226I C-RAF, we cannot determine whether Q335H C-RAF and E478K C-RAF are somatic mutations or rare human polymorphisms.However, because Q335H C-RAF and E478K C-RAF lack significantly elevated basal kinase activity and are not transforming, it is possible that they could be rare polymorphisms that predispose their carriers to specific forms of cancer; further studies are under way to examine this possibility.
It may be surprising that A-RAF and C-RAF mutations in cancer are so rare because both isoforms can be converted into transforming agents in experimental systems, typically by removing their NH 2 -terminal regulatory domains ( for review, see ref. 24).Indeed, a chromosomal inversion that essentially creates just this type of agent in B-RAF has been identified in a single case of human thyroid cancer linked to the Chernobyl nuclear accident (25).However, the genetic approach that we use cannot identify large deletions, gene inversions, or translocations, and thus it is possible that they were present in our samples but went undetected.Another surprise is that previous experimental  approaches have been used to induce transforming point mutations in full-length C-RAF, most of which are outside the kinase domain and a surprisingly large number are within CR2 (26)(27)(28), and yet we did not detect any corresponding mutations in our samples.Thus, although it is possible to create oncogenic versions of C-RAF, in practice mutations in human cancer seem to be almost exclusively in B-RAF.This suggests that either the rates of mutation of the RAF genes differ significantly or the regulatory networks of the different isoforms differ in such a manner as to select against A-RAF and C-RAF mutations.
Notwithstanding this reasoning, our data provide another rational explanation of why C-RAF mutations are rare in human cancer.We show that the V492E substitution activates C-RAF f45fold and over 1,000-fold when the N-region is charged.Similarly, V600E activates B-RAF f500-fold, unless the N-region is neutralized, then this decreases to f35-fold.Clearly, the V492/ V600 mutations cooperate with N-region charge to activate the RAF proteins and, working in conjunction, they act to convert C-RAF into an oncogene.Similar results are seen with the E478K mutation, demonstrating the importance of the N-region in revealing the transforming potential of the mutant C-RAF proteins.Presumably in the absence of this N-region charge, even if C-RAF mutations did occur in cancer, they would not enhance its activity sufficiently to convert C-RAF into an oncogene and, hence, they do not provide an advantage and so are not selected.Alternatively, if they occurred coincident with a second event, they may even stimulate excessive signaling and actually antagonize tumor progression.Presumably, the same holds true for A-RAF.
Although the N-region clearly plays an important role in augmenting the response of the RAF proteins to the mutants, this is not the only region that seems to reveal the oncogenic potential of B-RAF.We note that when the N-regions of E586K B-RAF and V600E B-RAF are neutralized, they retain some transforming activity.We have previously shown that the basal kinase activity of B-RAF is considerably higher than that of C-RAF (8) and it is clear that E586K B-RAF and V600E B-RAF have even higher basal kinase activities.We show here that C-RAF must be activated over 10,000-fold to achieve the same level of activity as E586K B-RAF, and yet this is still only f25% of the activity of V600E B-RAF.Furthermore, although DD,V492E C-RAF is over 1,000-fold more active than WT C-RAF, this is still only 2% to 5% of the activity of V600E B-RAF.Presumably, this explains why AAAA,V600E B-RAF is still transforming: its activity is about 2,500-fold above that of WT C-RAF.Thus, although the N-region is clearly important, it is not the only factor contributing to the elevated basal kinase activity of B-RAF.Further analysis of the differences between these proteins is warranted and it does not seem to be simply due to the fact that C-RAF cannot be activated to the same level as B-RAF, as shown with DD,E478K C-RAF.
We conclude that it is the key differences between B-RAF and C-RAF that regulate the different levels of basal kinase activities that account for the fact that whereas B-RAF mutations occur in f8% of cancers, C-RAF mutations are rare and only occur in specific cellular contexts.Finally, we note that our data could be interpreted to suggest that approaches to target the kinase responsible for phosphorylating the N-region of B-RAF would not provide effective anticancer therapies because residual transforming is activity retained.However, if agents such as these were combined with agents that directly target oncogenic B-RAF kinase activity, they could be of considerable value.

Figure 1 .
Figure 1.Amino acid changes in C-RAF.A, schematic of C-RAF.Light shaded boxes, CR1, CR2, and CR3; dark shaded boxes, N-region and the activation segment (Act.Seg. ).The positions of the four mutations identified in C-RAF in the cancer cell lines are also indicated.B, alignment of the amino acid sequences for specific regions of A-RAF, B-RAF, and C-RAF from humans (Hs ), mice (Mm), chickens (Gg), Xenopus (Xe), zebra fish (Dr ), Drosophila melanogaster (Dm), and C. elegans (Ce ).Mutations present in C-RAF in human cancer; bold, conserved in the other species.Gray box, N-region.

Figure 2 .
Figure 2. Characterization of P207S C-RAF, V226I C-RAF, and Q335H C-RAF.A, basal kinase activity; B, G12V RAS-activated kinase activity.Columns, mean from one assay done in triplicate; bars, SD.Similar results were obtained in at least three experiments.C, Western blot for C-RAF, ppMEK, and total MEK in COS cells expressing C-RAF or the indicated mutants.Oncogenic RAS was included where indicated.D, NIH 3T3 transformation assay.Transformation efficiency of NIH 3T3 cells by C-RAF (WT ) or the indicated mutants.V600E B-RAF is included as a positive control.Columns, mean of three independent determinations; bars, SD.Right, representative results from one assay.

Figure 3 .
Figure 3. RAS-dependent phosphorylation of Q335H C-RAF is impaired but its activation by mutant B-RAF is unaltered.A, Western blot for C-RAF and pS338 in COS cells expressing WT C-RAF or Q335H C-RAF.Where indicated, oncogenic RAS was coexpressed.B, Western blot for pS338 and total C-RAF in COS cells expressing C-RAF or the indicated mutants.Where indicated, oncogenic RAS was coexpressed.C, G12V RAS-and L597V B-RAF-stimulated C-RAF kinase activities.Columns, mean from one assay done in triplicate; bars, SD.Similar results were obtained in at least two experiments.

Figure 4 .
Figure 4. E478K C-RAF has elevated kinase activity but is not an oncogene.A, basal and G12V RAS-stimulated C-RAF kinase activities.Columns, mean from one assay done in triplicate; bars, SD.Activities are expressed as fold activation with respect to the wild-type protein.B, Western blot for C-RAF, ppMEK, and total MEK in COS cells expressing C-RAF or the indicated mutants.C, NIH 3T3 transformation assay.Transformation efficiency of NIH 3T3 cells by C-RAF or B-RAF (WT ) or the indicated mutants.Columns, mean of three independent assays; bars, SD.Right, representative results from one assay.D, C-RAF kinase activity; E, B-RAF kinase activity.Columns, mean from a single assay done in triplicate; bars, SD.Similar results were obtained in at least three independent experiments.

Figure 5 .
Figure 5. C-RAF is not activated by the V492E mutation and is not transforming.A, C-RAF kinase activity.Columns, mean from one assay done in triplicate; bars, SD.Activities are expressed as fold activation with respect to the wild-type protein and similar results were obtained in at least three experiments.B, Western blot for C-RAF, ppMEK, and total MEK in COS cells expressing C-RAF or V492E C-RAF.Where indicated, G12V RAS was coexpressed.C, NIH 3T3 transformation assay.Transformation efficiency of NIH 3T3 cells by C-RAF or B-RAF (WT ) or the indicated mutants.Columns, mean of three independent assays; bars, SD.Right, representative results from one assay.

Figure 6 .
Figure 6.V492E C-RAF becomes activated and transforming when negative charge is introduced at the N-region.A, basal and, where indicated, RAS-stimulated kinase activities.Columns, mean from one assay done in triplicate; bars, SD.Activities are expressed as fold activation with respect to the wild-type protein and similar results were obtained in at least three experiments.B, Western blot for C-RAF, ppMEK, and total MEK in COS cells expressing C-RAF or the specified mutants.Where indicated, G12V RAS was coexpressed.C, NIH 3T3 transformation assay.Transformation efficiency of NIH 3T3 cells by WT C-RAF or WT B-RAF or the indicated mutants.Columns, mean of three independent assays; bars, SD.Right, representative results from one assay.

Figure 7 .
Figure 7.The kinase activity of V600E B-RAF is impaired by neutralization of N-region charge but it is still transforming.A, B-RAF kinase activity.Columns, mean from one assay done in triplicate; bars, SD.Activities are expressed as fold activation with respect to WT B-RAF and similar results were obtained in at least three experiments.B, Western blot for B-RAF, ppMEK, and total MEK in COS cells expressing wild-type B-RAF or the specified mutants.C, NIH 3T3 transformation assay.Transformation efficiency of NIH 3T3 cells by WT B-RAF or the indicated mutants.Columns, mean of three independent assays; bars, SD.Right, representative results from one assay.

Table 1 .
C-RAF mutations in human cancer cell lines NOTE: Nucleotide changes and the corresponding amino acid changes are shown for individual cell lines.The statuses of RAS and B-RAF (amino acid changes only) in these cells are also presented.