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Conferring specificity on the ubiquitous Raf/MEK signalling pathway

Conferring specificity on the ubiquitous Raf/MEK signalling pathway British Journal of Cancer (2004) 90, 283 – 288 & 2004 Cancer Research UK All rights reserved 0007 – 0920/04 $25.00 www.bjcancer.com Minireview Conferring specificity on the ubiquitous Raf/MEK signalling pathway ,1 1,2 E O’Neill and W Kolch 1 2 Beatson Institute for Cancer Research, Switchback Road, Glasgow G61 1BD, UK; Institute of Biomedical and Life Sciences, Sir Henry Wellcome Functional Genomics Facility, University of Glasgow, Glasgow G12 8QQ, UK The Raf-MEK-ERK signalling pathway controls fundamental cellular processes including proliferation, differentiation and survival. It remains enigmatic how this pathway can reliably convert a myriad of extracellular stimuli in specific biological responses. Recent results have shown that the Raf family isoforms A-Raf, B-Raf and Raf-1 have different physiological functions. Here we review how Raf isozyme diversity contributes to the specification of functional diversity, in particular regarding the role of Raf isozymes in cancer. British Journal of Cancer (2003) 90, 283 – 288. doi:10.1038/sj.bjc.6601488 www.bjcancer.com & 2004 Cancer Research UK Keywords: Raf; MAPK; signalling; substrate; activation; isoforms Raf first came to the fore as a retroviral oncogene, v-Raf or v-Mil, requires points of crosstalk where these contextual signals are which could induce tumours in mice and chickens, respectively. integrated. In such a scenario, the exact kinetics and duration of Raf-1, the cognate proto-oncogene, is widely expressed and ERK signalling will play a major role. Such fine tuning can be became the most intensely studied Raf family member. The other adjusted by the differential response of upstream activators to two family members, A-Raf and B-Raf, feature a more restricted external cues. There may also be yet unrecognised branchpoints expression. A-Raf is mainly expressed in urogenital tissues and B- that distribute signals into other downstream pathways. These Raf expression is highest in neuronal tissues, testis and mechanisms are not mutually exclusive, and recent discoveries haematopoetic cells, although recent experiments suggest that support all of these possibilities. In this review, we will briefly the expression of A-Raf and B-Raf at low levels is much more discuss this evidence. widely spread (reviewed by Kolch, 2000). Interest in Raf proteins surged upon identification of their role as direct effectors of Ras. Ras has suffered oncogenic mutations in RAF GENE KNOCKOUTS nearly 30% of all human cancers. All the three Raf family members share the biochemical properties of binding to Ras and to The most convincing case for isozyme-specific functions of the phosphorylate and activate MEK (reviewed by Kolch, 2000; Avruch three Raf family genes was made by knockout studies in mice. The et al, 2001). Currently, MEK is the only generally acknowledged ablation of the A-raf gene (Pritchard et al, 1996) results in substrate of Raf kinases. MEK phosphorylates and activates ERK. neurological defects. In an inbred background, mice die 7–21 days While Raf and MEK appear restricted to only one class of post partum from megacolon caused by a defect in visceral substrates, ERK musters more than 70 substrates including nuclear neurons that control bowel contractions. In an outbred back- transcription factors (Lewis et al, 1998). This led to the perception ground, A-raf mice survive to adulthood, but still feature of a linear pathway where Ras funnels a great variety of defects in proprioception and abnormal movements. This extracellular cues into a three-tiered kinase module, Raf-MEK- phenotype resembles Neurotrophin-3 (NT-3)-deficient mice ERK, which relies on ERK to dispense the signal to various (Kirstein and Farinas, 2002). NT-3 also promotes the survival of substrates. An obvious problem with this view is how to rationalise visceral neurons. These observations would place A-Raf in a NT-3- that this pathway controls many and diverse fundamental cellular mediated neuronal survival pathway. The lack of A-Raf did not functions including proliferation, differentiation, transformation affect the regulation of ERK in mouse embryonic fibroblasts and apoptosis. (MEFs) or the transformation of these cells, suggesting that these The specificity of biological responses can be encoded in a functions are compensated for by the other Raf family members number of ways. Specificity can be achieved by compartmentalisa- (Mercer et al, 2002). tion, where the accessibility to upstream activators and down- Raf-1-deficient mice die in midgestation, due to widespread stream substrates is regulated by subcellular localisation. The apoptosis throughout the embryo (Huser et al, 2001; Mikula et al, response can also depend on the cellular context, such as the 2001). The penetrance of the phenotype was more severe in inbred expression of other proteins and activity of other pathways. This than in outbred backgrounds. Surprisingly, ERK activation by growth factors was not compromised in Raf-1 MEFs, pre- *Correspondence: Dr E O’Neill; E-mail: [email protected] sumably due to compensation by B-Raf. Even more surprisingly, a Received 19 August 2003; revised 14 October 2003; accepted 15 Raf-1 mutant that is refractory to growth factor stimulation could October 2003 rescue the knockout phenotype, resulting in viable and apparently Specificity of Raf/MEK signalling E O’Neill and W Kolch normal mice (Huser et al, 2001). Overall, the Raf-1 phenotype In contrast, B-Raf is activated directly by Ras binding. B-Raf also resembles K-Ras as well as Fas-ligand transgenic mice. These features a phosphomimetic aspartate in place of the residue observations suggest that Raf-1 is a main effector of K-Ras, and corresponding to Y341, and is constitutively phosphorylated at the that a major task of Raf-1 is to restrain the proapoptotic function S338 equivalent (Mason et al, 1999). Thus, B-Raf appears to have of Fas signalling. Indeed, Raf-1 MEFs exhibited increased shortcircuited several of the events required for the activation of sensitivity to Fas-induced apoptosis (Huser et al, 2001; Mikula Raf-1. This is consistent with B-Raf possessing a much higher et al, 2001). Much needed detailed mechanistic investigations are specific activity towards MEK than Raf-1. This could explain why just beginning. The cause of the anaemia in Raf-1 animals was B-raf can fully compensate for the loss of Raf-1 or A-Raf in the traced back to the elevation of caspase-1 activity that accelerated knockout cells pertaining to the regulation of ERK. erythroid differentiation to a point where erythroid progenitor In addition, B-Raf can be activated by Rap1 (York et al, 1998), a cells became depleted (Kolbus et al, 2002). All these observations Ras-related protein, which was originally described as a suppressor point to effectors distinct from MEK and ERK mediating Raf-1’s of Ras transformation. This inhibitory function seems to be due to antiapoptotic role. the ability of Rap1 to sequester Raf-1 in inactive complexes. As this B-raf mice (Wojnowski et al, 1997) provided the first genetic requires Rap1 overexpression, it is unclear whether this function of evidence for a role of a Raf isoform in the regulation of apoptosis. Rap1 is physiological. Rap1 is activated by numerous growth These mice die in midgestation, due to haemorrhage caused by factors and appears to be a signal transducer in its own right (Bos massive apoptosis of endothelial cells. However, the interpretation et al, 2001). Again, it has been disputed whether B-Raf is a of these data is confounded by the uncertainty whether the physiological Rap1 effector (Bos et al, 2001), but at least under knockout strategy has abolished the expression of all the B-raf certain conditions it can promote PC12 cell differentiation by splice forms. Nevertheless, the very different phenotypes of activating B-Raf. The higher activity of B-Raf ensures the sustained the Raf knockout mice indicate that Raf isoforms have different activation of ERK that is required for neuronal differentiation of biological functions. B-Raf emerges as the main regulator of PC12 cells. However, there is also differential regulation down- the MEK-ERK pathway, while Raf-1 and A-Raf seem to provide stream through crosstalk with the cAMP signalling system. The ERK-independent apoptosis protection in different tissues. cAMP-dependent protein kinase PKA phosphorylates and inhibits This conclusion is likely too simplistic, but can serve as a Raf-1. In contrast, cAMP induces the activation of B-Raf by useful working hypothesis for the dissection of Raf isoform activation of Rap1 (York et al, 1998). Thus, cAMP should interfere function. with ERK activation in cells where only Raf-1 is expressed, but An elegant biochemical study of the contribution of Raf-1 and B- promote ERK activation in cells where B-Raf is coexpressed. Raf to B-cell receptor (BCR) signalling showed that Raf-1 and B- Switching from one Raf isoform with low MEK kinase activity to Raf are activated with different kinetics and cooperate in many another with high activity could serve to fine-tune the activation downstream responses (Brummer et al, 2002). DT40 B-cells were dynamics of ERK, in order to determine a specific biological engineered so that either gene could be deleted individually, or response (Figure 1). A recent study has shown how ERK activation both together. Raf-1 was dispensable for BCR-mediated ERK kinetics can be converted into differential responses (Murphy et al, activation, while the ablation of B-Raf caused a reduced and 2002). A transient activation of ERK will induce the expression of shortened activity of ERK. The Raf-1/B-raf double knockout Fos by phosphorylating the Elk and Sap transcription factors. As resulted in an almost complete loss of ERK activation and severely the Fos protein is very unstable, the impact on a transcriptional reduced expression of the downstream transcriptional targets c- response is limited. However, sustained ERK signalling results in Fos and Egr-1. Only the activation of nuclear factor of activated T the activation of Rsk, which phosphorylates and stabilises Fos, cells, NFAT, was mainly dependent on B-Raf. The molecular basis producing a robust transcriptional activation. for the cooperation between Raf-1 and B-Raf is not yet understood. It could rely on complementation at the level of downstream substrates, including specific differences in activation kinetics. It RAF ISOFORMS IN CANCER also could be related to the ability of Raf-1 to heterodimerise with B-Raf. The heterodimer, conceivably, could have different signal- Raf-1 is the cellular proto-oncogene homologue of v-Raf. v-Raf ling properties than either individual protein. corresponds to the Raf-1 kinase domain. Indeed, the deletion of the regulatory domain converts Raf-1 into an oncogene, termed BXB (Heidecker et al, 1990). However, genetic alterations of Raf-1 in human tumours have not been found. Lung tumour cell lines RAF ISOFORM REGULATION AND FUNCTION often overexpress Raf-1, and a transgenic mouse model has been All the three Raf isoforms contain a Ras-binding site (RBD) in the developed where Raf gene expression is targeted to the lungs by regulatory domain at the N-terminus, and can consequently be use of a tissue-specific promoter (Rapp et al, 2003). BXB activated by Ras GTPases. The RBD selectively binds to activated overexpression rapidly induced numerous well-differentiated, GTP-loaded Ras, but the effects are different. Ras binding does not noninvasive adenomas. Surprisingly, the overexpression of Raf-1, activate Raf-1 directly, but seems to serve to translocate Raf-1 from which is nontransforming in cell culture models, also resulted in the cytosol to the membrane, where subsequent activation events the formation of adenomas with the same histological phenotype. occur (Dhillon and Kolch, 2002). These comprise a complex series These tumours were fewer and delayed, and, in contrast to BXB, of events starting with the dephosphorylation of S259, which induced adenomas did not feature elevated ERK activity. These allows phosphorylation of S338 and possibly Y341 as well as two findings suggest that Raf-1 contributes to different aspects of sites in the activation loop. These modifications work together not malignancy, including ERK-independent processes and those only to determine the quantity of the signalling output, but also which escape in vitro experimentation. Crossing the BXB maybe even the quality. Raf-1 S259 mutant can activate ERK to a transgenic mice with bcl-2 knockout mice did not change the similar extent as the v-Raf oncogene, yet fails to transform the tumour phenotype, but retarded tumour development mainly due cells, suggesting that the quality of the downstream signal is to an increase in the rate of apoptosis. In contrast, the concomitant different depending on the upstream mode of activation (Dhillon loss of p53 accelerated tumour growth and also changed the et al, 2003). Rather little is known about the regulation of A-Raf, histological composition from cuboid cells to papillary tumours but it seems to be regulated in a similar way as Raf-1 yet binds to with large columnar epithelial cells. Despite occasional bronchiolar Ras more weakly and also is a weaker MEK kinase (Marais et al, invasion, no metastasis was observed. These phenotypes resemble 1997). human atypical adenomatous lung hyperplasia, which may be the British Journal of Cancer (2004) 90(2), 283 – 288 & 2004 Cancer Research UK Specificity of Raf/MEK signalling E O’Neill and W Kolch EGF EGF Transient ERK Proliferation Raf−1 Ras Ras Raf activity MEK ERK cAMP NGF Rap B−raf Differentiation Sustained ERK activity Figure 1 PC12 cell model of neuronal differentiation. This model shows how a biological response is specified by the kinetics and duration of ERK activity, which is achieved through the combinatorial integration of activating different Raf isoforms and crosstalk with the cAMP signalling system. PC12 cells differentiate in response to the nerve growth factor (NGF), but proliferate in response to the epidermal growth factor (EGF). Both growth factors utilise the Raf/MEK/ERK pathway. The biological response is determined by the duration of ERK signalling. Sustained ERK activation results in neuronal differentiation. The sustenance of ERK activity is caused by the B-Raf isoform, which is activated preferentially by NGF. Differentiation is further enhanced by activation of cAMP signalling, which inhibits Raf-1, but promotes B-Raf activity. initial lesion on the way to lung adenocarcinoma (Mori et al, 2001). of Raf-1 in this very complicated manner? A provocative Thus, studies to validate the role of Raf-1 in human lung cancer are possibility is that MEK is not the main relevant substrate of Raf- eagerly awaited. In particular, it may be interesting to examine the 1, or that these post-translational modifications serve as docking role of B-Raf. platforms to assemble functionally different Raf-1 complexes. B-Raf is mutated in 66% of melanomas and a smaller number of The interpretation of yet unknown Raf-1 substrates is in line other prevalent cancers such as colon tumours. While the with the phenotype of the Raf-1 mice which succumb to activation of Raf-1 requires a complex series of events, the deregulated apoptosis despite an apparently normal regulation of activation of B-Raf is much simpler to achieve, explaining why B- ERK. There is also a growing body of circumstantial evidence that Raf is a preferred target for mutational activation in human not all functions of Raf-1 are dependent on its ability to activate cancers (Mercer and Pritchard, 2003). The main mutation is MEK (Hindley and Kolch, 2002). Obviously, the existence of Raf V599E, which introduces a negative charge into the activation isoform-specific substrates would neatly explain isoform diversity. loop. This phosphomimetic mutation suffices to deregulate B-Raf A number of alternative Raf-1 substrates have been described, but activity and convert it into an oncogene (Davies et al, 2002). In the none has been unequivocally validated yet. One presumably Raf meantime, a flurry of studies has established that B-Raf is indeed isoform-specific substrate is the retinoblastoma protein (Rb). Rb mutated in a great variety of cancers albeit, with the exception of phosphorylation is required to traverse the G1–S-phase boundary thyroid carcinoma, B-Raf mutation has a rather low incidence of the cell cycle. Although Rb is classically viewed as a target for (Mercer and Pritchard, 2003). Most of these tumour types are cyclin D- and E-dependent cell cycle kinases, other kinases may known to have Ras mutations, but interestingly, Ras and B-Raf contribute to its inactivation. Raf-1 has been reported to promote mutations usually do not coincide in the same tumour. This the inactivation of Rb by directly phosphorylating it. Rb observation provides strong genetic evidence that B-Raf is a crucial phosphorylation was dependent on binding to the 25 N-terminal effector for Ras-mediated tumourigenesis; however, the exact amino acids of Raf-1. This stretch is unique and hence Rb should molecular mechanism is still obscure. Besides the prevalent V599E be a Raf-1 isoform-specific target (Wang et al, 1998). mutation, which enhances kinase activity, there are three other However, there is also the possibility that Raf isoforms convey classes of mutations whose functional consequences are less clear specificity through conveying differential activation kinetics on (Mercer and Pritchard, 2003). One class affects the Akt phosphor- their common substrate MEK. The PC12 cell paradigm has been ylation sites in B-Raf, which have been implicated in the inhibition discussed above. Studies in IL-3-dependent haematopoetic cell of B-raf kinase activity. Obviously, these mutations would only lines showed that the A-Raf kinase domain abrogates growth factor activate B-raf in cells where its activity is restrained through Akt. dependence more efficiently than the Raf-1 or B-Raf kinase Another class of mutations that leads to modest activation is found domains. However, in all cases, this process was MEK dependent in the ATP-binding glycine-rich loop. Yet another type alters the (Hoyle et al, 2000). conserved DFG motif at the base of the activation loop. Such Yet another level of specificity could be achieved through the mutations usually incapacitate the kinase activity. These mutations differential activation of MEK and ERK isoforms. Both MEK and are rare, but challenge the view that the oncogenic conversion of B- ERK feature two isoforms in mammalian cells. A common Raf is only due to the chronic hyperstimulation of the MEK-ERK assumption is that they are functionally equivalent. However, pathway. A plausible explanation is that B-Raf has a role that is their evolutionary conservation suggests that they exert nonre- independent of its kinase activity, for instance, as a scaffolding dundant functions. This is proven by the phenotype of MEK and protein or as a dominant-negative mutant, which sequesters ERK knockout mice. Knocking out MEK-1 results in an embryonic proteins that prevent transformation. lethal phenotype which is similar, but not identical to the Raf-1 knockout (Giroux et al, 1999). This confirms that MEK-1 is a physiologically relevant target of Raf-1, but also points to a more complicated scenario where signals diversify from MEK isoforms. DIVERSIFICATION AT THE SUBSTRATE LEVEL Indeed, in Hela cells, A-Raf selectively activated MEK-1 in B-Raf appears to be the main activator of the MEK-ERK pathway response to EGF, whereas Raf-1 activated both MEK-1 and MEK- (Huser et al, 2001; Mikula et al, 2001), and the simplicity of B-Raf 2 (Wu et al, 1996). In a similar vein, the ablation of ERK-1 does not activation rationalises why B-raf is a preferred target for oncogenic compromise viability and causes rather subtle changes in the mutation. However, it also leaves a puzzle. Why does nature go to functions of T-cells and memory. In contrast, knocking out ERK-2 such great lengths to regulate the comparably poor kinase activity is embryonic lethal (Saba-El-Leil et al, 2003). At present, the & 2004 Cancer Research UK British Journal of Cancer (2004) 90(2), 283 – 288 p14 p14 p14 p14 p14 p14 K K K K Ksr s s s srrrr K K K K K Ksr s s s s srrrrr Specificity of Raf/MEK signalling E O’Neill and W Kolch molecular basis for this diversification is unclear, as usually both therapeutic intervention. Interfering with KSR expression or MEK and ERK isoforms are coregulated. A potential answer could function would be expected to impede tumour growth, but leave be provided by differential subcellular compartmentalisation and normal cells unscathed. complex formation. Another example is MP-1, a small scaffold that ties MEK and ERK together. MP-1 also binds to p14, an endosomal protein, which targets the MEK/ERK/MP-1 signalling complex to late endosomes. The downregulation of p14 or MP-1 protein levels SIGNAL DIVERSIFICATION THROUGH SCAFFOLDING diminished ERK activation and the induction of Elk-1-dependent PROTEINS AND SUBCELLULAR LOCALISATION reporter gene expression (Teis et al, 2002). These results suggest Signalling through this pathway is regulated by protein interac- that the Raf/MEK/ERK pathway is organised in spatially distinct tions that serve to connect activators with effectors, as well as signalling complexes. It remains to be shown whether this target them to different subcellular localisations (Kolch, 2000). The correlates with functional diversity. An attractive possibility is binding of Raf kinases to Ras translocates Raf to the membrane that the spatial segregation could determine selective interactions compartment, where activation ensues. The artificial targeting to with upstream activators and downstream effectors (Figure 2). the plasma membrane suffices to partially activate Raf-1. Ha-Ras is For instance, a fraction of Raf-1 was found at the mitochondria not only activated at the cell membrane, but also on endomem- and the artificial targeting of activated Raf-1 to mitochondria branes, resulting in a differential interaction with downstream inhibited apoptosis. Curiously, mitochondrial Raf-1 did not targets. Tethering Ha-Ras to the Golgi preferentially activated JNK, activate the MEK-ERK pathway, but rather phosphorylated and while Ha-Ras targeted to the endoplasmic reticulum stimulated inactivated BAD, a proapoptotic protein (Wang et al, 1996). ERK and Akt activities (Chiu et al, 2002). This observation Unfortunately, BAD was not confirmed as a direct Raf-1 substrate, suggests that the quality of the downstream signal is determined by and the search for the physiological substrate of mitochondrial the subcellular localisation of Ras, which by implication regulates Raf-1 is still ongoing. Interestingly, A-Raf was also detected in rat the interaction with downstream effectors. liver mitochondria. In contrast to Raf-1, which is loosely and This theme is also encountered further downstream in the peripherally associated, A-raf appears to be a true mitochondrial pathway. The scaffolding protein KSR constitutively binds to MEK. protein. It interacts with the putative mitochondrial import In response to mitogenic stimulation, the KSR/MEK complex is proteins hTOM and hTIM, and is found in the intermembrane recruited from the cytosol to the cell membrane, where it can now space and the matrix (Yuryev et al, 2000). The function of A-Raf at interact with activated Raf-1 and ERK to facilitate the signal flux the mitochondria is unknown. However, given that A-Raf is a very through the kinase module Raf-MEK-ERK (Muller et al, 2001). poor MEK kinase, an alternative mitochondrial substrate seems Gene knockout experiments in the worm C. elegans, which like plausible. mammals has two KSR genes, has revealed overlapping as well as These results also demonstrate that the identification of some specific functions for the two KSR genes. However, when interaction partners is a viable strategy to unravel new functional both genes were removed, ERK activation was severely compro- connections. A recent systematic study used the N-terminal mised and the phenotype was similar to disabling the let-60 Ras regulatory domains of Raf-1 and A-Raf, respectively, as baits in gene (Ohmachi et al, 2002). Knocking out KSR1 in mice did not an exhaustive yeast two-hybrid screen (Yuryev and Wennogle, result in any gross abnormalities, although ERK activation in 2003). In total, 20 different proteins were identified, including response to growth factors was modestly attenuated and the T-cell several novel interaction partners. Half of these proteins exhibited response to antigen was impeded. Remarkably, however, the isoform selectivity, six proteins binding to A-raf and four to Raf-1. KSR1 mice were significantly less susceptible to developing Even more surprisingly, this selectivity was encoded by the mammary tumours when crossed to a transgenic tumour-prone cysteine-rich zinc-binding domain (CRD). This domain is essential strain (Nguyen et al, 2002). This opens a new opportunity for for activation by Ras, although it is not the primary Ras-binding Ras Ras Ras Ras Ras Ras Ras Ras Raf Raf Raf Raf Raf Raf Raf Raf Raf ME ME ME ME MEK K K K K MEK MEK MEK MEK MEK Raf Raf Raf Late MP1 MP1 MP1 MP1 MP1 MP1 ERK ERK ERK ERK ERK endosome ERK ERK ERK ERK ERK MEK MEK MEK MEK ERK ERK ERK mitoch mitoch mitoch mitoch mitochondri ondri ondri ondri ondria a a a a Raf Raf Raf Raf Specific ? ? ? ? responses Figure 2 Different signalling complexes and subcellular compartmentalisation can generate diverse cellular responses. See text for details. British Journal of Cancer (2004) 90(2), 283 – 288 & 2004 Cancer Research UK Specificity of Raf/MEK signalling E O’Neill and W Kolch site (Avruch et al, 2001). These data pinpoint the CRD as a major of Raf-1 by phosphorylation of S338 and Raf-1 translocation to hub for isoform selective protein interactions. They also predict mitochondria. In contrast, VEGF-mediated survival is MEK the existence of a great number of distinct Raf signalling dependent, does not translocate Raf-1 to mitochondria, and uses complexes, as the small size of the CRD makes simultaneous phosphorylation of Y341 to activate Raf-1. This example shows interactions unlikely. Clearly, a future challenge will be to unravel how the mode of activation could specify downstream signalling the composition of multiprotein signalling complexes in situ and through differential subcellular localisation. Unveiling this combi- characterise the dynamics of the interactions. The recent progress natorial complexity will be the next great challenge for modern in proteomics and the improvement of imaging techniques for biology. monitoring protein interactions at subcellular resolution and in This task will also have enormous repercussions on drug real time will allow us to tackle these questions. development. Potent Raf and MEK inhibitors have been tested in clinical trials. In particular, the Raf inhibitor BAY 43-9006 was well tolerated, showing good efficacy as a single agent and in CONCLUSION combination therapies (Hotte and Hirte, 2002). However, the realisation that one protein can have different functions in The number of our genes is too small to account for the complexity different cellular contexts will warrant new strategies for drug of biological functions. Thus, the cell employs the same proteins in design. Target validation will rely on understanding these different contexts and imposes specificity through combinatorial networks, which will only be possible at a systems biology level mechanisms. Using the Raf/MEK/ERK signalling pathway as requiring quantitative biology combined with massive in silico paradigm, we have highlighted some of these mechanisms simulation. We also will need to develop appropriate screens for including differential protein interactions, subcellular compart- new categories of targets, which may include protein interactions mentalisation, different modes of activation, and differential and subcellular distribution. Future drug development will rely on targeting of downstream effectors. A recent study elegantly vigorous basic research in these areas and interdisciplinary demonstrates how the cell orchestrates this repertoire of mechan- collaboration to translate the findings into applications. isms (Alavi et al, 2003). In endothelial cells, vascular endothelial growth factor (VEGF) protects from apoptosis caused by serum starvation and DNA-damaging drugs, whereas basic fibroblast growth factor (bFGF) prevents the apoptosis induced by death ACKNOWLEDGEMENTS receptor stimulation. Both pathways employ Raf-1, yet use different modes of activation and different downstream effectors. 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Conferring specificity on the ubiquitous Raf/MEK signalling pathway

O'Neill, E; Kolch, W
British Journal of Cancer , Volume 90 (2) – Jan 20, 2004
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Springer Journals
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Copyright © 2004 by The Author(s)
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Biomedicine; Biomedicine, general; Cancer Research; Epidemiology; Molecular Medicine; Oncology; Drug Resistance
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0007-0920
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1532-1827
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10.1038/sj.bjc.6601488
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Abstract

British Journal of Cancer (2004) 90, 283 – 288 & 2004 Cancer Research UK All rights reserved 0007 – 0920/04 $25.00 www.bjcancer.com Minireview Conferring specificity on the ubiquitous Raf/MEK signalling pathway ,1 1,2 E O’Neill and W Kolch 1 2 Beatson Institute for Cancer Research, Switchback Road, Glasgow G61 1BD, UK; Institute of Biomedical and Life Sciences, Sir Henry Wellcome Functional Genomics Facility, University of Glasgow, Glasgow G12 8QQ, UK The Raf-MEK-ERK signalling pathway controls fundamental cellular processes including proliferation, differentiation and survival. It remains enigmatic how this pathway can reliably convert a myriad of extracellular stimuli in specific biological responses. Recent results have shown that the Raf family isoforms A-Raf, B-Raf and Raf-1 have different physiological functions. Here we review how Raf isozyme diversity contributes to the specification of functional diversity, in particular regarding the role of Raf isozymes in cancer. British Journal of Cancer (2003) 90, 283 – 288. doi:10.1038/sj.bjc.6601488 www.bjcancer.com & 2004 Cancer Research UK Keywords: Raf; MAPK; signalling; substrate; activation; isoforms Raf first came to the fore as a retroviral oncogene, v-Raf or v-Mil, requires points of crosstalk where these contextual signals are which could induce tumours in mice and chickens, respectively. integrated. In such a scenario, the exact kinetics and duration of Raf-1, the cognate proto-oncogene, is widely expressed and ERK signalling will play a major role. Such fine tuning can be became the most intensely studied Raf family member. The other adjusted by the differential response of upstream activators to two family members, A-Raf and B-Raf, feature a more restricted external cues. There may also be yet unrecognised branchpoints expression. A-Raf is mainly expressed in urogenital tissues and B- that distribute signals into other downstream pathways. These Raf expression is highest in neuronal tissues, testis and mechanisms are not mutually exclusive, and recent discoveries haematopoetic cells, although recent experiments suggest that support all of these possibilities. In this review, we will briefly the expression of A-Raf and B-Raf at low levels is much more discuss this evidence. widely spread (reviewed by Kolch, 2000). Interest in Raf proteins surged upon identification of their role as direct effectors of Ras. Ras has suffered oncogenic mutations in RAF GENE KNOCKOUTS nearly 30% of all human cancers. All the three Raf family members share the biochemical properties of binding to Ras and to The most convincing case for isozyme-specific functions of the phosphorylate and activate MEK (reviewed by Kolch, 2000; Avruch three Raf family genes was made by knockout studies in mice. The et al, 2001). Currently, MEK is the only generally acknowledged ablation of the A-raf gene (Pritchard et al, 1996) results in substrate of Raf kinases. MEK phosphorylates and activates ERK. neurological defects. In an inbred background, mice die 7–21 days While Raf and MEK appear restricted to only one class of post partum from megacolon caused by a defect in visceral substrates, ERK musters more than 70 substrates including nuclear neurons that control bowel contractions. In an outbred back- transcription factors (Lewis et al, 1998). This led to the perception ground, A-raf mice survive to adulthood, but still feature of a linear pathway where Ras funnels a great variety of defects in proprioception and abnormal movements. This extracellular cues into a three-tiered kinase module, Raf-MEK- phenotype resembles Neurotrophin-3 (NT-3)-deficient mice ERK, which relies on ERK to dispense the signal to various (Kirstein and Farinas, 2002). NT-3 also promotes the survival of substrates. An obvious problem with this view is how to rationalise visceral neurons. These observations would place A-Raf in a NT-3- that this pathway controls many and diverse fundamental cellular mediated neuronal survival pathway. The lack of A-Raf did not functions including proliferation, differentiation, transformation affect the regulation of ERK in mouse embryonic fibroblasts and apoptosis. (MEFs) or the transformation of these cells, suggesting that these The specificity of biological responses can be encoded in a functions are compensated for by the other Raf family members number of ways. Specificity can be achieved by compartmentalisa- (Mercer et al, 2002). tion, where the accessibility to upstream activators and down- Raf-1-deficient mice die in midgestation, due to widespread stream substrates is regulated by subcellular localisation. The apoptosis throughout the embryo (Huser et al, 2001; Mikula et al, response can also depend on the cellular context, such as the 2001). The penetrance of the phenotype was more severe in inbred expression of other proteins and activity of other pathways. This than in outbred backgrounds. Surprisingly, ERK activation by growth factors was not compromised in Raf-1 MEFs, pre- *Correspondence: Dr E O’Neill; E-mail: [email protected] sumably due to compensation by B-Raf. Even more surprisingly, a Received 19 August 2003; revised 14 October 2003; accepted 15 Raf-1 mutant that is refractory to growth factor stimulation could October 2003 rescue the knockout phenotype, resulting in viable and apparently Specificity of Raf/MEK signalling E O’Neill and W Kolch normal mice (Huser et al, 2001). Overall, the Raf-1 phenotype In contrast, B-Raf is activated directly by Ras binding. B-Raf also resembles K-Ras as well as Fas-ligand transgenic mice. These features a phosphomimetic aspartate in place of the residue observations suggest that Raf-1 is a main effector of K-Ras, and corresponding to Y341, and is constitutively phosphorylated at the that a major task of Raf-1 is to restrain the proapoptotic function S338 equivalent (Mason et al, 1999). Thus, B-Raf appears to have of Fas signalling. Indeed, Raf-1 MEFs exhibited increased shortcircuited several of the events required for the activation of sensitivity to Fas-induced apoptosis (Huser et al, 2001; Mikula Raf-1. This is consistent with B-Raf possessing a much higher et al, 2001). Much needed detailed mechanistic investigations are specific activity towards MEK than Raf-1. This could explain why just beginning. The cause of the anaemia in Raf-1 animals was B-raf can fully compensate for the loss of Raf-1 or A-Raf in the traced back to the elevation of caspase-1 activity that accelerated knockout cells pertaining to the regulation of ERK. erythroid differentiation to a point where erythroid progenitor In addition, B-Raf can be activated by Rap1 (York et al, 1998), a cells became depleted (Kolbus et al, 2002). All these observations Ras-related protein, which was originally described as a suppressor point to effectors distinct from MEK and ERK mediating Raf-1’s of Ras transformation. This inhibitory function seems to be due to antiapoptotic role. the ability of Rap1 to sequester Raf-1 in inactive complexes. As this B-raf mice (Wojnowski et al, 1997) provided the first genetic requires Rap1 overexpression, it is unclear whether this function of evidence for a role of a Raf isoform in the regulation of apoptosis. Rap1 is physiological. Rap1 is activated by numerous growth These mice die in midgestation, due to haemorrhage caused by factors and appears to be a signal transducer in its own right (Bos massive apoptosis of endothelial cells. However, the interpretation et al, 2001). Again, it has been disputed whether B-Raf is a of these data is confounded by the uncertainty whether the physiological Rap1 effector (Bos et al, 2001), but at least under knockout strategy has abolished the expression of all the B-raf certain conditions it can promote PC12 cell differentiation by splice forms. Nevertheless, the very different phenotypes of activating B-Raf. The higher activity of B-Raf ensures the sustained the Raf knockout mice indicate that Raf isoforms have different activation of ERK that is required for neuronal differentiation of biological functions. B-Raf emerges as the main regulator of PC12 cells. However, there is also differential regulation down- the MEK-ERK pathway, while Raf-1 and A-Raf seem to provide stream through crosstalk with the cAMP signalling system. The ERK-independent apoptosis protection in different tissues. cAMP-dependent protein kinase PKA phosphorylates and inhibits This conclusion is likely too simplistic, but can serve as a Raf-1. In contrast, cAMP induces the activation of B-Raf by useful working hypothesis for the dissection of Raf isoform activation of Rap1 (York et al, 1998). Thus, cAMP should interfere function. with ERK activation in cells where only Raf-1 is expressed, but An elegant biochemical study of the contribution of Raf-1 and B- promote ERK activation in cells where B-Raf is coexpressed. Raf to B-cell receptor (BCR) signalling showed that Raf-1 and B- Switching from one Raf isoform with low MEK kinase activity to Raf are activated with different kinetics and cooperate in many another with high activity could serve to fine-tune the activation downstream responses (Brummer et al, 2002). DT40 B-cells were dynamics of ERK, in order to determine a specific biological engineered so that either gene could be deleted individually, or response (Figure 1). A recent study has shown how ERK activation both together. Raf-1 was dispensable for BCR-mediated ERK kinetics can be converted into differential responses (Murphy et al, activation, while the ablation of B-Raf caused a reduced and 2002). A transient activation of ERK will induce the expression of shortened activity of ERK. The Raf-1/B-raf double knockout Fos by phosphorylating the Elk and Sap transcription factors. As resulted in an almost complete loss of ERK activation and severely the Fos protein is very unstable, the impact on a transcriptional reduced expression of the downstream transcriptional targets c- response is limited. However, sustained ERK signalling results in Fos and Egr-1. Only the activation of nuclear factor of activated T the activation of Rsk, which phosphorylates and stabilises Fos, cells, NFAT, was mainly dependent on B-Raf. The molecular basis producing a robust transcriptional activation. for the cooperation between Raf-1 and B-Raf is not yet understood. It could rely on complementation at the level of downstream substrates, including specific differences in activation kinetics. It RAF ISOFORMS IN CANCER also could be related to the ability of Raf-1 to heterodimerise with B-Raf. The heterodimer, conceivably, could have different signal- Raf-1 is the cellular proto-oncogene homologue of v-Raf. v-Raf ling properties than either individual protein. corresponds to the Raf-1 kinase domain. Indeed, the deletion of the regulatory domain converts Raf-1 into an oncogene, termed BXB (Heidecker et al, 1990). However, genetic alterations of Raf-1 in human tumours have not been found. Lung tumour cell lines RAF ISOFORM REGULATION AND FUNCTION often overexpress Raf-1, and a transgenic mouse model has been All the three Raf isoforms contain a Ras-binding site (RBD) in the developed where Raf gene expression is targeted to the lungs by regulatory domain at the N-terminus, and can consequently be use of a tissue-specific promoter (Rapp et al, 2003). BXB activated by Ras GTPases. The RBD selectively binds to activated overexpression rapidly induced numerous well-differentiated, GTP-loaded Ras, but the effects are different. Ras binding does not noninvasive adenomas. Surprisingly, the overexpression of Raf-1, activate Raf-1 directly, but seems to serve to translocate Raf-1 from which is nontransforming in cell culture models, also resulted in the cytosol to the membrane, where subsequent activation events the formation of adenomas with the same histological phenotype. occur (Dhillon and Kolch, 2002). These comprise a complex series These tumours were fewer and delayed, and, in contrast to BXB, of events starting with the dephosphorylation of S259, which induced adenomas did not feature elevated ERK activity. These allows phosphorylation of S338 and possibly Y341 as well as two findings suggest that Raf-1 contributes to different aspects of sites in the activation loop. These modifications work together not malignancy, including ERK-independent processes and those only to determine the quantity of the signalling output, but also which escape in vitro experimentation. Crossing the BXB maybe even the quality. Raf-1 S259 mutant can activate ERK to a transgenic mice with bcl-2 knockout mice did not change the similar extent as the v-Raf oncogene, yet fails to transform the tumour phenotype, but retarded tumour development mainly due cells, suggesting that the quality of the downstream signal is to an increase in the rate of apoptosis. In contrast, the concomitant different depending on the upstream mode of activation (Dhillon loss of p53 accelerated tumour growth and also changed the et al, 2003). Rather little is known about the regulation of A-Raf, histological composition from cuboid cells to papillary tumours but it seems to be regulated in a similar way as Raf-1 yet binds to with large columnar epithelial cells. Despite occasional bronchiolar Ras more weakly and also is a weaker MEK kinase (Marais et al, invasion, no metastasis was observed. These phenotypes resemble 1997). human atypical adenomatous lung hyperplasia, which may be the British Journal of Cancer (2004) 90(2), 283 – 288 & 2004 Cancer Research UK Specificity of Raf/MEK signalling E O’Neill and W Kolch EGF EGF Transient ERK Proliferation Raf−1 Ras Ras Raf activity MEK ERK cAMP NGF Rap B−raf Differentiation Sustained ERK activity Figure 1 PC12 cell model of neuronal differentiation. This model shows how a biological response is specified by the kinetics and duration of ERK activity, which is achieved through the combinatorial integration of activating different Raf isoforms and crosstalk with the cAMP signalling system. PC12 cells differentiate in response to the nerve growth factor (NGF), but proliferate in response to the epidermal growth factor (EGF). Both growth factors utilise the Raf/MEK/ERK pathway. The biological response is determined by the duration of ERK signalling. Sustained ERK activation results in neuronal differentiation. The sustenance of ERK activity is caused by the B-Raf isoform, which is activated preferentially by NGF. Differentiation is further enhanced by activation of cAMP signalling, which inhibits Raf-1, but promotes B-Raf activity. initial lesion on the way to lung adenocarcinoma (Mori et al, 2001). of Raf-1 in this very complicated manner? A provocative Thus, studies to validate the role of Raf-1 in human lung cancer are possibility is that MEK is not the main relevant substrate of Raf- eagerly awaited. In particular, it may be interesting to examine the 1, or that these post-translational modifications serve as docking role of B-Raf. platforms to assemble functionally different Raf-1 complexes. B-Raf is mutated in 66% of melanomas and a smaller number of The interpretation of yet unknown Raf-1 substrates is in line other prevalent cancers such as colon tumours. While the with the phenotype of the Raf-1 mice which succumb to activation of Raf-1 requires a complex series of events, the deregulated apoptosis despite an apparently normal regulation of activation of B-Raf is much simpler to achieve, explaining why B- ERK. There is also a growing body of circumstantial evidence that Raf is a preferred target for mutational activation in human not all functions of Raf-1 are dependent on its ability to activate cancers (Mercer and Pritchard, 2003). The main mutation is MEK (Hindley and Kolch, 2002). Obviously, the existence of Raf V599E, which introduces a negative charge into the activation isoform-specific substrates would neatly explain isoform diversity. loop. This phosphomimetic mutation suffices to deregulate B-Raf A number of alternative Raf-1 substrates have been described, but activity and convert it into an oncogene (Davies et al, 2002). In the none has been unequivocally validated yet. One presumably Raf meantime, a flurry of studies has established that B-Raf is indeed isoform-specific substrate is the retinoblastoma protein (Rb). Rb mutated in a great variety of cancers albeit, with the exception of phosphorylation is required to traverse the G1–S-phase boundary thyroid carcinoma, B-Raf mutation has a rather low incidence of the cell cycle. Although Rb is classically viewed as a target for (Mercer and Pritchard, 2003). Most of these tumour types are cyclin D- and E-dependent cell cycle kinases, other kinases may known to have Ras mutations, but interestingly, Ras and B-Raf contribute to its inactivation. Raf-1 has been reported to promote mutations usually do not coincide in the same tumour. This the inactivation of Rb by directly phosphorylating it. Rb observation provides strong genetic evidence that B-Raf is a crucial phosphorylation was dependent on binding to the 25 N-terminal effector for Ras-mediated tumourigenesis; however, the exact amino acids of Raf-1. This stretch is unique and hence Rb should molecular mechanism is still obscure. Besides the prevalent V599E be a Raf-1 isoform-specific target (Wang et al, 1998). mutation, which enhances kinase activity, there are three other However, there is also the possibility that Raf isoforms convey classes of mutations whose functional consequences are less clear specificity through conveying differential activation kinetics on (Mercer and Pritchard, 2003). One class affects the Akt phosphor- their common substrate MEK. The PC12 cell paradigm has been ylation sites in B-Raf, which have been implicated in the inhibition discussed above. Studies in IL-3-dependent haematopoetic cell of B-raf kinase activity. Obviously, these mutations would only lines showed that the A-Raf kinase domain abrogates growth factor activate B-raf in cells where its activity is restrained through Akt. dependence more efficiently than the Raf-1 or B-Raf kinase Another class of mutations that leads to modest activation is found domains. However, in all cases, this process was MEK dependent in the ATP-binding glycine-rich loop. Yet another type alters the (Hoyle et al, 2000). conserved DFG motif at the base of the activation loop. Such Yet another level of specificity could be achieved through the mutations usually incapacitate the kinase activity. These mutations differential activation of MEK and ERK isoforms. Both MEK and are rare, but challenge the view that the oncogenic conversion of B- ERK feature two isoforms in mammalian cells. A common Raf is only due to the chronic hyperstimulation of the MEK-ERK assumption is that they are functionally equivalent. However, pathway. A plausible explanation is that B-Raf has a role that is their evolutionary conservation suggests that they exert nonre- independent of its kinase activity, for instance, as a scaffolding dundant functions. This is proven by the phenotype of MEK and protein or as a dominant-negative mutant, which sequesters ERK knockout mice. Knocking out MEK-1 results in an embryonic proteins that prevent transformation. lethal phenotype which is similar, but not identical to the Raf-1 knockout (Giroux et al, 1999). This confirms that MEK-1 is a physiologically relevant target of Raf-1, but also points to a more complicated scenario where signals diversify from MEK isoforms. DIVERSIFICATION AT THE SUBSTRATE LEVEL Indeed, in Hela cells, A-Raf selectively activated MEK-1 in B-Raf appears to be the main activator of the MEK-ERK pathway response to EGF, whereas Raf-1 activated both MEK-1 and MEK- (Huser et al, 2001; Mikula et al, 2001), and the simplicity of B-Raf 2 (Wu et al, 1996). In a similar vein, the ablation of ERK-1 does not activation rationalises why B-raf is a preferred target for oncogenic compromise viability and causes rather subtle changes in the mutation. However, it also leaves a puzzle. Why does nature go to functions of T-cells and memory. In contrast, knocking out ERK-2 such great lengths to regulate the comparably poor kinase activity is embryonic lethal (Saba-El-Leil et al, 2003). At present, the & 2004 Cancer Research UK British Journal of Cancer (2004) 90(2), 283 – 288 p14 p14 p14 p14 p14 p14 K K K K Ksr s s s srrrr K K K K K Ksr s s s s srrrrr Specificity of Raf/MEK signalling E O’Neill and W Kolch molecular basis for this diversification is unclear, as usually both therapeutic intervention. Interfering with KSR expression or MEK and ERK isoforms are coregulated. A potential answer could function would be expected to impede tumour growth, but leave be provided by differential subcellular compartmentalisation and normal cells unscathed. complex formation. Another example is MP-1, a small scaffold that ties MEK and ERK together. MP-1 also binds to p14, an endosomal protein, which targets the MEK/ERK/MP-1 signalling complex to late endosomes. The downregulation of p14 or MP-1 protein levels SIGNAL DIVERSIFICATION THROUGH SCAFFOLDING diminished ERK activation and the induction of Elk-1-dependent PROTEINS AND SUBCELLULAR LOCALISATION reporter gene expression (Teis et al, 2002). These results suggest Signalling through this pathway is regulated by protein interac- that the Raf/MEK/ERK pathway is organised in spatially distinct tions that serve to connect activators with effectors, as well as signalling complexes. It remains to be shown whether this target them to different subcellular localisations (Kolch, 2000). The correlates with functional diversity. An attractive possibility is binding of Raf kinases to Ras translocates Raf to the membrane that the spatial segregation could determine selective interactions compartment, where activation ensues. The artificial targeting to with upstream activators and downstream effectors (Figure 2). the plasma membrane suffices to partially activate Raf-1. Ha-Ras is For instance, a fraction of Raf-1 was found at the mitochondria not only activated at the cell membrane, but also on endomem- and the artificial targeting of activated Raf-1 to mitochondria branes, resulting in a differential interaction with downstream inhibited apoptosis. Curiously, mitochondrial Raf-1 did not targets. Tethering Ha-Ras to the Golgi preferentially activated JNK, activate the MEK-ERK pathway, but rather phosphorylated and while Ha-Ras targeted to the endoplasmic reticulum stimulated inactivated BAD, a proapoptotic protein (Wang et al, 1996). ERK and Akt activities (Chiu et al, 2002). This observation Unfortunately, BAD was not confirmed as a direct Raf-1 substrate, suggests that the quality of the downstream signal is determined by and the search for the physiological substrate of mitochondrial the subcellular localisation of Ras, which by implication regulates Raf-1 is still ongoing. Interestingly, A-Raf was also detected in rat the interaction with downstream effectors. liver mitochondria. In contrast to Raf-1, which is loosely and This theme is also encountered further downstream in the peripherally associated, A-raf appears to be a true mitochondrial pathway. The scaffolding protein KSR constitutively binds to MEK. protein. It interacts with the putative mitochondrial import In response to mitogenic stimulation, the KSR/MEK complex is proteins hTOM and hTIM, and is found in the intermembrane recruited from the cytosol to the cell membrane, where it can now space and the matrix (Yuryev et al, 2000). The function of A-Raf at interact with activated Raf-1 and ERK to facilitate the signal flux the mitochondria is unknown. However, given that A-Raf is a very through the kinase module Raf-MEK-ERK (Muller et al, 2001). poor MEK kinase, an alternative mitochondrial substrate seems Gene knockout experiments in the worm C. elegans, which like plausible. mammals has two KSR genes, has revealed overlapping as well as These results also demonstrate that the identification of some specific functions for the two KSR genes. However, when interaction partners is a viable strategy to unravel new functional both genes were removed, ERK activation was severely compro- connections. A recent systematic study used the N-terminal mised and the phenotype was similar to disabling the let-60 Ras regulatory domains of Raf-1 and A-Raf, respectively, as baits in gene (Ohmachi et al, 2002). Knocking out KSR1 in mice did not an exhaustive yeast two-hybrid screen (Yuryev and Wennogle, result in any gross abnormalities, although ERK activation in 2003). In total, 20 different proteins were identified, including response to growth factors was modestly attenuated and the T-cell several novel interaction partners. Half of these proteins exhibited response to antigen was impeded. Remarkably, however, the isoform selectivity, six proteins binding to A-raf and four to Raf-1. KSR1 mice were significantly less susceptible to developing Even more surprisingly, this selectivity was encoded by the mammary tumours when crossed to a transgenic tumour-prone cysteine-rich zinc-binding domain (CRD). This domain is essential strain (Nguyen et al, 2002). This opens a new opportunity for for activation by Ras, although it is not the primary Ras-binding Ras Ras Ras Ras Ras Ras Ras Ras Raf Raf Raf Raf Raf Raf Raf Raf Raf ME ME ME ME MEK K K K K MEK MEK MEK MEK MEK Raf Raf Raf Late MP1 MP1 MP1 MP1 MP1 MP1 ERK ERK ERK ERK ERK endosome ERK ERK ERK ERK ERK MEK MEK MEK MEK ERK ERK ERK mitoch mitoch mitoch mitoch mitochondri ondri ondri ondri ondria a a a a Raf Raf Raf Raf Specific ? ? ? ? responses Figure 2 Different signalling complexes and subcellular compartmentalisation can generate diverse cellular responses. See text for details. British Journal of Cancer (2004) 90(2), 283 – 288 & 2004 Cancer Research UK Specificity of Raf/MEK signalling E O’Neill and W Kolch site (Avruch et al, 2001). These data pinpoint the CRD as a major of Raf-1 by phosphorylation of S338 and Raf-1 translocation to hub for isoform selective protein interactions. They also predict mitochondria. In contrast, VEGF-mediated survival is MEK the existence of a great number of distinct Raf signalling dependent, does not translocate Raf-1 to mitochondria, and uses complexes, as the small size of the CRD makes simultaneous phosphorylation of Y341 to activate Raf-1. This example shows interactions unlikely. Clearly, a future challenge will be to unravel how the mode of activation could specify downstream signalling the composition of multiprotein signalling complexes in situ and through differential subcellular localisation. Unveiling this combi- characterise the dynamics of the interactions. The recent progress natorial complexity will be the next great challenge for modern in proteomics and the improvement of imaging techniques for biology. monitoring protein interactions at subcellular resolution and in This task will also have enormous repercussions on drug real time will allow us to tackle these questions. development. Potent Raf and MEK inhibitors have been tested in clinical trials. In particular, the Raf inhibitor BAY 43-9006 was well tolerated, showing good efficacy as a single agent and in CONCLUSION combination therapies (Hotte and Hirte, 2002). However, the realisation that one protein can have different functions in The number of our genes is too small to account for the complexity different cellular contexts will warrant new strategies for drug of biological functions. Thus, the cell employs the same proteins in design. Target validation will rely on understanding these different contexts and imposes specificity through combinatorial networks, which will only be possible at a systems biology level mechanisms. Using the Raf/MEK/ERK signalling pathway as requiring quantitative biology combined with massive in silico paradigm, we have highlighted some of these mechanisms simulation. We also will need to develop appropriate screens for including differential protein interactions, subcellular compart- new categories of targets, which may include protein interactions mentalisation, different modes of activation, and differential and subcellular distribution. Future drug development will rely on targeting of downstream effectors. A recent study elegantly vigorous basic research in these areas and interdisciplinary demonstrates how the cell orchestrates this repertoire of mechan- collaboration to translate the findings into applications. isms (Alavi et al, 2003). In endothelial cells, vascular endothelial growth factor (VEGF) protects from apoptosis caused by serum starvation and DNA-damaging drugs, whereas basic fibroblast growth factor (bFGF) prevents the apoptosis induced by death ACKNOWLEDGEMENTS receptor stimulation. Both pathways employ Raf-1, yet use different modes of activation and different downstream effectors. 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British Journal of Cancer – Springer Journals

Published: Jan 20, 2004

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