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Molecular Biology, Pathobiology and Genetics |
1 The Institute of Cancer Research, Signal Transduction Team, Cancer Research UK Centre of Cell and Molecular Biology, London, United Kingdom and 2 The Wellcome Trust Sanger Institute, Hinxton, United Kingdom
Requests for reprints: Richard Marais, Signal Transduction Team, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, United Kingdom. Phone: 44-20-7878-3856; Fax: 44-20-7352-3299; E-mail: richard.marais{at}icr.ac.uk.
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Abstract |
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8% 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. |
Introduction |
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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 338SSY341Y 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 8% of human cancers (5, 9). Importantly, a glutamic acid substitution for the valine at codon 600 (V600) accounts for 90% of the B-RAF mutations, demonstrating extraordinary selection for this genetic lesion. V600EB-RAF is activated 500-fold; it stimulates constitutive MEK-ERK signaling in cells and transforms fibroblasts and melanocytes (6, 9–12). 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.
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Results |
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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). E586KB-RAF has 130-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 (G12VRAS; Fig. 2A and B). The basal kinase activities of P207SC-RAF and V266IC-RAF are similar to that of WTC-RAF and their activation by G12VRAS is also similar to that of WTC-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).
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130-fold more active than WTB-RAF (6). Importantly, whereas E586KB-RAF stimulates strong constitutive MEK-ERK signaling in COS cells (6, 14), MEK activation by E478KC-RAF is weak (Fig. 4B); furthermore, whereas E586KB-RAF transforms NIH 3T3 cells, E478KC-RAF does not (Fig. 4C). Remarkably, however, whereas WTC-RAF is activated 100-fold by G12VRAS, E478KC-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 WTC-RAF. Thus, E478KC-RAF is hypersensitive to activation by oncogenic RAS, suggesting that this mutant can be strongly activated by secondary signals.
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3-fold; not obvious in Fig. 4D) but, strikingly, it synergizes with E478K (DD,E478KC-RAF) to activate C-RAF over 10,000-fold, a level that is commensurate with the activity of E586KB-RAF (Fig. 4D). DDC-RAF also synergizes with E478KC-RAF to activate MEK in COS cells (Fig. 4B) and to transform NIH 3T3 cells (Fig. 4C). We also did the reverse experiment, neutralizing the charge within the N-region of E586KB-RAF by substituting S446, S447, D448, and D449 with alanines (AAAA,E586KB-RAF). Introducing these substitutions into E586KB-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,E586KB-RAF) and the D448A/D449A (SSAA,E586KB-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 E586KB-RAF. Finally, we show that the transforming activity of E586KB-RAF is significantly reduced when the N-region charge is disrupted (Fig. 4C). We conclude that E478KC-RAF is strongly activated by mutations that introduce a constitutive negative charge into its N-region whereas the activity of E586KB-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. V492EC-RAF is 45-fold more active than WTC-RAF (Fig. 5A), a level of activation that contrasts strongly with the 500-fold activation seen with the corresponding mutation in B-RAF (V600EB-RAF; ref. 6). Furthermore, whereas V600EB-RAF stimulates strong constitutive MEK/ERK signaling in mammalian cells (9) and is transforming (Fig. 2D), V492EC-RAF only stimulates weak MEK activity in COS cells (Fig. 5B) and does not transform NIH 3T3 cells (Fig. 5C).
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15-fold reduction in kinase activity but we note that the kinase is still 35-fold more active than WTB-RAF (Fig. 7A). Importantly, AAAA,V600EB-RAF still activates MEK in COS cells (Fig. 6B) and transforms NIH 3T3 cells (Fig. 7C) albeit at reduced levels.
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Discussion |
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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 G12VRAS, 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 L597VB-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.5
We have been unable to study the biological function of Q335HC-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 (16–21). 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 1% 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), 30% of the inactive mutants occur in cancers that also harbor RAS mutations and we have argued that the D594 mutants could act in a dominant-negative manner to suppress excessive RAS-MEK signaling (5). Thus, it is intriguing that in addition to the Q335HC-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 E478KC-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 E478KC-RAF is only apparent in some cell contexts. In common with P207SC-RAF and V226IC-RAF, we cannot determine whether Q335HC-RAF and E478KC-RAF are somatic mutations or rare human polymorphisms. However, because Q335HC-RAF and E478KC-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 NH2-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 (26–28), 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 45-fold and over 1,000-fold when the N-region is charged. Similarly, V600E activates B-RAF 500-fold, unless the N-region is neutralized, then this decreases to 35-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 E586KB-RAF and V600EB-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 E586KB-RAF and V600EB-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 E586KB-RAF, and yet this is still only 25% of the activity of V600EB-RAF. Furthermore, although DD,V492EC-RAF is over 1,000-fold more active than WTC-RAF, this is still only 2% to 5% of the activity of V600EB-RAF. Presumably, this explains why AAAA,V600EB-RAF is still transforming: its activity is about 2,500-fold above that of WTC-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,E478KC-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 8% 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.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Footnotes |
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M. Garnett is currently at the Department of Oncology, Hutchison/Medical Research Council Research Centre, University of Cambridge, Hills Road, Cambridge CB2 2XZ, United Kingdom. C. Mason is currently at the Molecular Sciences Sareum Ltd., 2 Pampisford Park, London Road, Pampisford, Cambridge CB2 4EE, United Kingdom.
3 http://www.ncbi.nlm.nih.gov/entrez/. 
4 http://www.ncbi.nlm.nih.gov/.
5 M. Garnett and R. Marais, submitted for publication.
Received 5/16/05. Revised 7/11/05. Accepted 8/31/05.
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