Prospects & Overviews

Review essays

The Ras-ERK pathway: Understanding site-specic signaling provides hope of new anti-tumor therapies
n ez and Piero Crespo Fernando Calvo, Lorena Agudo-Iba

Recent discoveries have suggested the concept that intracellular signals are the sum of multiple, site-specied subsignals, rather than single, homogeneous entities. In the context of cancer, searching for compounds that selectively block subsignals essential for tumor progression, but not those regulating house-keeping functions, could help in producing drugs with reduced side effects compared to compounds that block signaling completely. The Ras-ERK pathway has become a paradigm of how space can differentially shape signaling. Today, we know that Ras proteins are found in different plasma membrane microdomains and endomembranes. At these localizations, Ras is subject to site-specic regulatory mechanisms, distinctively engaging effector pathways and switching-on diverse genetic programs to generate different biological responses. The Ras effector pathway leading to ERKs activation is also under strict, space-related regulatory processes. These ndings may open a gate for aiming at the Ras-ERK pathway in a spatially restricted fashion, in our quest for new anti-tumor therapies. Keywords: Cancer; ERK; MAP kinases; Ras; Spatial regulation of signalling

Introduction
Signal transduction focuses on the molecular mechanisms whereby signals, received at the surface of the cell, are propagated throughout its interior. These switch on biochemical processes and genetic programs necessary for generating biological responses, such as proliferation, differentiation, and survival. Countless studies demonstrate that severe pathological conditions, cancer in particular, can arise when signaling pathways go awry. Thus, during the past decades, signaling intermediaries have been subject to an intense scrutiny, in an effort to identify among them targets for therapeutic intervention through which aberrant signals and their consequential pathological conditions could be forestalled. Among the legions of signaling molecules that have been under intense examination, Ras and its effector pathways have probably drawn the greatest attention. Ras proteins (H-, N-, and K-Ras) are key regulators of essential cellular processes. Their importance in cell physiology is highlighted by the dramatic results of their malfunction: Ras is the most frequent oncogene in human cancers, with mutational activation being detected in approximately 30% of human tumors. If we add to this gure the cases in which alterations are detected in components of Ras effector pathways, in a non-overlapping occurrence, in particular of the route leading to ERK activation (Fig. 1), the frequency nearly reaches 50%.[1] Added to this, the experimental evidence substantiating the importance of the Ras-ERK pathway in cancer initiation and progression is

DOI 10.1002/bies.200900155
a de Cantabria (IBBTEC), Consejo Instituto de Biomedicina y Biotecnolog cas (CSIC), IDICAN, Universidad de Superior de Investigaciones Cient Cantabria, Cantabria, Spain *Corresponding author: Piero Crespo E-mail: crespop@unican.es Abbreviations: ABV, Avian Bronchitis Virus; ER, Endoplasmic reticulum; ERK, Extracellularregulated Kinase; PM, Plasma Membrane; GAP, GTPase Activating Protein; GEF, Guanine Nucleotide Exchange Factor; GC, Golgi Complex; GPCR, G protein-coupled Receptor; MAPK, Mitogen-activated Protein Kinase; PLA2, Phospholipase A2; PKC, Protein Kinase C

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Figure 1. A simplied representation of the Ras-ERK pathway, including its main components. The frequency of activating mutations in different human neoplasias is shown.

duration and, subsequently, directly affects therapeutic efcacy. In light of these considerations: is the Ras-ERK pathway an example of too-sensitive-a-target for therapeutic intervention? Are there alternative ways to aim at Ras-ERK signals to enable the generation of more efcient while less toxic inhibitors? An opportunity may have arisen following recent discoveries that demonstrate the spatial segregation of Ras-ERK signals. We now know that Ras is present in various membrane systems where its signals are regulated in distinct manners. Similarly, ERK signals unfold in different environments, like the nucleus and the cytoplasm, where they are inuenced by a broad collection of site-specic regulators. Do these spatially restricted subsignals contribute to the same extent and in the same way to tumorigenesis? Are site-specic Ras-ERK subsignals similar in normal versus tumor cells? Could we exploit some difference for targeting tumor cells more efciently, while sparing normal ones? Here, we review the current knowledge on the compartmentalization of Ras-ERK signals, with emphasis on the potential applications to the design of new generations of anti-tumor drugs.

Review essays

overwhelming: activated mutants of Ras, Raf, and MEK have been shown to induce malignant transformation in many cell types. Studies using genetic and pharmacological interference of the Ras-ERK pathway have demonstrated that its signals are essential for the maintenance of the transformed phenotype in diverse tumor-derived cells,[1, 2] and sophisticated animal models have endorsed the importance of this pathway for tumorigenesis in vivo.[3] Thus, it is hardly surprising that this signaling cascade has attracted enormous attention for antineoplastic intervention. Colossal efforts have been devoted, both by academia and industry, rst to nd drugable targets among its constituents and, subsequently, to generate compounds aimed at such signaling intermediaries. Aiming at components of the Ras-ERK pathway as an antitumor strategy is an old story not devoid of disappointments. Initially, substantial efforts were devoted to the inhibition of activated Ras proteins, mainly by the use of farnesyltransferase inhibitors. However, clinical trials with these have been largely unsuccessful (for a review see ref. 4). With Ras out of the limelight, the downstream kinases Raf and MEK remained attractive candidates. But, considering the central role played by ERK in the regulation of physiological proliferation, there has always been concern that an effective blockade of ERK may just prove too toxic for normal cells, particularly those forming epithelia with a high turn-over. Preclinical studies suggest that complete and continuous suppression of ERK activation would be required for maximal therapeutic effects.[5] But, clinical trials with several Raf and MEK inhibitors have revealed toxic effects such as diarrhea, skin rash, fatigue, visual disturbances, etc., in protocols where ERK phosphorylation is reduced signicantly, although far from completely and continuously.[5] In extreme cases, such as the one for PD0325901, this has prompted its discontinuation (for extensive reviews see ref. [6] and [7]). Toxicity is a factor that imposes severe limitations on treatment dosage and

Ras: An actor on many stages

It has long been known that Ras proteins must associate to membranes to be functional. Initially, Ras was thought to act exclusively at the plasma membrane (PM).[8] More recently, thanks to the pioneering work of M. Philips, it was demonstrated that Ras was also present and active in endomembranes like the endoplasmic reticulum (ER) and the Golgi complex (GC).[9, 10] The three Ras isoforms were also detected in endosomes[11, 12] and mitochondria[1315] (for a review see ref. 16). Further complexity was introduced when it was found that, within the PM, Ras segregated at distinct microdomains: H- and N-Ras were present in cholesterol-rich domains, also known as lipid rafts, and in disordered membrane domains, whereas K-Ras was only detected in the latter.[17, 18] Importantly, Ras sublocalization appeared to depend on its activation status: inactive, GDP-bound, H-Ras associated predominantly to lipid rafts, translocating to disordered membrane when GTP-loaded.[17] Such a widespread distribution is possible mainly due to the post-translational processing experienced by Ras proteins. These are synthesized as hydrophilic proteins terminating in a CAAX motif, which undergoes a series of modications including prenylation, proteolysis, and carboxyl methylation (for a review see ref. 19). The cysteine in the CAAX box is recognized by farnesyltransferases that catalyze the addition of a farnesyl isoprenoid,[20] which permits a transient association to the ER. For stable membrane association Ras requires a second signal: H-Ras, N-Ras, and K-Ras4A are further modied by one or two palmitates.[21] This modication takes place at the GC.[22] K-Ras4B second signal consists of a polybasic region, rich in lysine residues, that exerts a strong electrostatic interaction with membrane phospholipids.[23] Importantly, Ras proteins are no longer thought to be stationary, but rather they are known to translocate between

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Figure 2. Ras acylation cycle. H- and N-Ras are palmitoylated at the GC by acyl-transferases of the DHCC9/GCP16 families. From there, they are transported, via vesicular transport, to their nal destinations at the PM. There, they are depalmitoylated, mainly by APT-1 acyl-thioesterase, and recycled back to the GC probably escorted by some chaperone that insulates the hydrophobic isoprenyl moiety.

nanoclusters: antagonizing Galectin-1 results in the displacement of H-Ras from the PM and in the inhibition of Ras biological activity.[37] Galectin-1 not only behaves as a key scaffold for the formation of H-Ras nanoclusters, it also functions as a molecular chaperone that contributes to H-Ras trafcking, by returning depalmitoylated H-Ras to the GC.[38]

cellular compartments. Whereas farnesylation is fairly stable, palmitoylation is rapidly reversible.[24] N- and H-Ras undergo a palmitoylation/depalmitoylation cycle, whereby palmitoylated forms at the PM are released following depalmitoylation, and trafc, via retrograde transport, to the GC. Once there, Ras proteins are repalmitoylated and sent back to the PM by vesicular transport (Fig. 2).[25, 26] Non-palmitoylated K-Ras4B also trafcs back and forth from the PM, although by other mechanisms: phosphorylation of K-Ras4B by PKC promotes its release from the PM and its transfer to mitochondria.[27] Calmodulin binding to K-Ras4B also dislodges it from the PM, facilitating its translocation to the GC and to early endosomes.[28] The presence of Ras in endosomes, and in the endocytic route in general, is signicant. Active H-Ras is internalized together with EGF receptors after stimulation.[29] Interestingly, Ras association to endosomes exhibits isoform specicity: whereas N- and H-Ras establish stable interactions with the endosomal compartment, K-Ras does not.[11, 30, 31] These differences have been attributed to the modication of N- and H-Ras, but not of K-Ras, by ubiquitination.[31] At the PM, Ras proteins partition into microdomains referred to as nanoclusters. Formation of these nanostructures requires the interaction with Galectins.[32] Following stimulation, Galectin-1 is recruited to the PM in response to H-Ras activation,[33] inducing the formation of activated H-Ras/Galectin-1 complexes;[34] these become enriched in cholesterol-independent microdomains.[35] Galectin-3 functions in a similar way, but specically for K-Ras.[36] These ancillary proteins are critical for the formation of

Ras activation and activities: When site matters

Not unexpectedly, the presence of Ras at multiple sites has functional consequences that result both from particularities in the way Ras activation is regulated at distinct localizations and from quantitative and qualitative variability in the way Ras engages its effector pathways. Originally, guanine nucleotide exchange factors (GEFs) were thought to activate Ras exclusively at the PM.[39] Later, it was found that Ras was also switched-on at different endomembranes and by distinct GEFs. For example, Ras is activated by Ras-GRP GEFs at the GC,[40, 41] whereas SOS and Ras-GRF GEFs function at the ER.[42] The way Ras is activated is also inuenced by, for example, Galectin-3 that inhibits Ras-GRP4 to divert incoming signals away from H- and N-Ras nanoclusters, thereby redirecting signaling to K-Ras nanoclusters.[43] Site-specic effects have also been observed for the deactivation of Ras by GTPase activating proteins (GAPs). Whereas calcium stimulates Ras activation at the PM using Ras-GRF in epithelial cells,[42] in lymphocytes, it promotes Ras activation at the GC through Ras-GRP1 and triggers PM Ras inactivation by the calcium-responsive GAP CAPRI.[41] Another ancillary protein, Annexin A6, promotes Ras inactivation in non-raft PM microdomains and in PM-derived endosomes, by forming a complex therein with p120 GAP.[44] A consequence of the site-specic activity of GEFs, GAPs, and other regulatory proteins is that such compartmentalization

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results in a remarkable variability in Ras signal outputs. Thus, Ras activation at the PM is fast and transient, whereas at endomembranes[45, 46] and at endosomes[47] is slow and sustained. Galectin-1, by stabilizing H-Ras nanoclusters, leads to enhanced recruitment of effectors and greater signal output.[34] In addition, formation of Ras nanoclusters transforms analog signal input into digital output,[48] which may be related to the potentiation of cellular transformation by Galectin-1.[37, 38] Importantly, Ras sublocalization markedly inuences which effector pathways are activated and how intensively. Probably, this has a lot to do with the abundance and availability of effector molecules at different sites. For example, both B-Raf and C-Raf interact with membrane phospholipids. Some of these, like phosphatidylethanolamine and phosphatidylinositol, inhibit their kinase activity.[49] It is possible that the amount of Raf available for activation and its degree of activation could be regulated by the phospholipid composition of the membranes where Ras is acting. Similarly, the presence of regulatory proteins like RKIP, a phosphatidylethanolamine-binding Raf inhibitor,[50] could also be dependent on the membrane composition. Most of what we know about Ras compartmentalized functions, we have learned by the use of articially tethered constructs. Seminal studies by Mark Philips showed that H-RasV12 conned to the ER, activated ERK, PI3K, and JNK. ERK activation was also observed when H-RasV12 was tethered to the GC.[45] A later study from our laboratory utilized the same strategy to study effector usage at PM microdomains as well: H-RasV12 at lipid rafts and at the ER efciently activated ERK, PI3K, and Ral-GDS; at bulk membrane it behaved similarly, although with lower PI3K activation.[51] H-Ras restricted to the GC, profusely activated Ral-GDS but not ERK. Even though some of these results may differ, probably due to experimental details, the concept of different localizations conferring variability in Ras effector usage has been rmly established. This notion also seems to hold at the nano scale: EGF stimulates Raf recruitment to K-Ras but not to H-Ras nanoclusters.[52] Effector usage can be inuenced by site-specic regulatory proteins, as exemplied by Galectins: Galectin-1 diverts H-Ras signals to Raf-1 at the expense of PI3K,[38] whereas Galectin-3 attenuates ERK activation without affecting PI3K activity.[36] Ras trafcking can also impact on effector utilization. Ubiquitination of H- and N-Ras promotes their association to endosomes, where reduced availability of Raf results in diminished ERK activation.[31] In Drosophila, a threshold of Ras ubiquitination is required to prevent excess ERK signaling,[53] an example of how Ras-ERK signaling can be regulated by connement to a specic compartment. Ultimately, such variability in the use of effector pathways results in marked differences in the transcription programs switched-on by Ras from each of its localizations.[54]

ultimately shape the resulting biological outputs. It has long been recognized that in PC12 cells, transient activation of the Ras-ERK pathway induces proliferation, whereas prolonged activation causes differentiation.[55] Such an effect must entail Ras trafcking, since conning the GTPase to lipid rafts severely inhibits sustained ERK activation and prevents PC12 differentiation.[56] By the use of articially tethered constructs, some Ras biological effects have been assigned to specic localizations. In agreement with Raf/ERK activation proles, Ras effectively supported proliferation and cellular transformation of murine broblasts from the ER, disordered membrane and lipid rafts, but not from GC.[51] A discrepancy exists with respect to Ras transforming potential at the GC,[45] probably due to differences in the GC tethers utilized, although in both studies transformation correlates with ERK activation. Ras tethered to the GC by the E1 ABV protein, was also unable to transform.[57] Furthermore, RKTG, a seven transmembrane protein localized at the GC, unproductively sequestered Raf into the GC, inhibiting ERK activation and PC12 differentiation,[58] also suggesting that the GC platform negatively regulates Ras-ERK signals. In agreement, in thymocytes, positive versus negative selection leads to proliferation and apoptosis respectively, both outcomes requiring Ras-ERK activation. Negative selection results from a strong PM Ras-ERK activation. Weaker antigens that induce positive selection generate a milder Ras-ERK signal at the GC.[59] Interestingly, in murine broblasts, only ER-tethered H-RasV12 could sustain survival in response to growth factor starvation.[51] N-Ras, which generates a potent anti-apoptotic signal,[14] is particularly enriched at the ER, implying that this endomembrane could be an important site for the regulation of survival. The ER is not likely to be the only source of such signals: phosphorylation of K-Ras by PKC promotes its rapid dissociation from the PM and its translocation to mitochondria, where it induces apoptosis.[27] This observation demonstrates that the same actor, K-Ras, at two different sites, PM versus mitochondria, can yield two diametrically opposed biological outcomes, proliferation or death.

Review essays

Some space for ERKs

Even though it may just be the tip of the iceberg, these data should be sufcient for abandoning the classical model of one, linear Ras pathway. In its place, we should envision several of these pathways, each at its own territory, each with its own regulators (Fig. 3). Such a notion does much to explain the diversity in outputs that Ras signals can achieve. It is important to realize that Ras localization is not the only source of spatial variability. Other components of the underlying cascades contribute in their own way. Such is the case for ERK 1 and 2 (ERKs) MAP kinases. We now know that ERKs are strictly regulated by multiple elements that modulate their localization and site-specic activities (Fig. 4). Under resting conditions, ERKs are found in the cytoplasm, as a consequence of their interaction with several types of cytoplasmic anchors. Once phosphorylated, ERKs lose their afnity for these partners and undergo a rapid relocalization to

The environment sets the response

The aforementioned differences in timing, intensity, and intermediaries that dene each site-specic Ras subsignal

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Figure 3. Ras compartmentalized functions and site-specic regulators. The isoforms present stably at each subcellular compartment are shown in blue. In purple, when presence is dependent on

modications: ub, ubiquitination; cal, calmodulin binding; P, phosphorylation. Ancillary proteins specically acting at particular sites are also depicted.

Figure 4. Compartmentalized regulation of ERKs. Scaffold proteins acting at specic sublocalizations are shown. ERKs functions at the cytoplasm are undertaken mainly as dimers whereas it translocates

to the nucleus as monomers. ERKs nuclear translocation can be prevented by proteins like PEA15, Bik, and DAPK, resulting in apoptosis.

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the nucleus, where they phosphorylate multiple nuclear proteins. In the nucleus, ERKs perform essential functions that regulate transcription, DNA replication, chromatin remodeling, and miRNA synthesis[60, 61] among others. However, ERKs extranuclear component is just as important. It has been estimated that half of the ERKs content remains in the cytoplasm after stimulation[62] and processes such as the formation of cell-matrix contacts,[63] adhesion,[64] endosomal trafc,[65] Golgi fragmentation,[66] and anti-apoptotic signaling[67] are dependent on ERK extranuclear activity. It fact, nearly half of the $180 proteins thus far identied as ERKs substrates, are non-nuclear proteins.[68] The nucleo-cytoplasmic shuttling of ERKs is exquisitely regulated. Some proteins, like PEA15, prevent ERKs nuclear translocation[69] and in doing so, promote ERK cytoplasmic activities, such as the modulation of integrin receptors,[70] while interfering with ERKs nuclear functions, like the promotion of proliferation.[71] For efcient nuclear translocation, ERKs require direct interaction with the nuclear pore complex,[72] a nuclear translocation signal within their insert domain[73] and the participation of nuclear shuttles like Importin-7[74] or Mxi2.[75] Interestingly, ERK functions within the nucleus may also be compartmentalized further: ERKs interact with lamin A at the nuclear envelope to promote rapid, mitogen-dependent AP-1 activation, by releasing cFos from its inhibitory interaction with lamin A.[76]

Recent ndings from our laboratory have put together space, scaffolds and substrates to help explain how scaffolds achieve spatial selectivity for ERKs signals. The microenvironment from which Ras signals emanate determines which substrates will be preferentially phosphorylated by activated ERKs, with such substrate specicity being governed by the participation of distinct scaffold proteins.[87] Specically, ERKs activated by Ras at the ER utilize Sef-1 to activate cPLA2. cPLA2 is also activated by ERKs in response to Ras signals coming from lipid rafts, but in this case the mediating scaffold is KSR1. KSR1 is not the only scaffold utilized by lipid raft-generated signals, since the phosphorylation of another ERK substrate, EGFr, is mediated by IQGAP. Another space-related regulatory mechanism that has been disclosed recently concerns ERK dimerization. ERKs are known to dimerize upon phosphorylation.[88] Initially, dimerization was proposed to be important for ERK nuclear import,[88] but it was later shown that ERKs nuclear inux occurs mainly in monomeric form,[89] leaving dimers orphans of a function. We have recently demonstrated that scaffold proteins serve as dimerization platforms in which ERK dimers are assembled. These scaffold-dimer complexes are essential for the ensuing interaction of ERKs with their cognate cytoplasmic substrates. In contrast, activation of ERKs nuclear substrates does not require the participation of scaffolds, in most cases, and is chiey undertaken by ERK monomers.[90] As such, dimerization and scaffolding appear to be essential processes for ERKs cytoplasmic but not nuclear signaling.

Review essays

A place for a scaffold

In addition to the kinase tiers, the ERK cascade includes other proteins that regulate the amplitude, intensity, and spatial specicity of its signals. Scaffold proteins serve all of these purposes. The main function of scaffolds is to bring together the members of the signaling cascade, forming a complex which helps in optimizing the signal. At the same time, they exclude other related components that operate in parallel cascades, insulating a MAPK module from undesired interferences.[77] In addition, scaffolds play important roles in the spatial selectivity of ERK signals: KSR1 acts preferentially upon ERK signals emanating from PM cholesterol-rich domains;[78] MP-1 regulates ERK in endosomes;[79] Sef at the GC;[80] and Paxillin at focal adhesions.[81] b-Arrestins are abundant in clathrin-coated pits.[82] They enhance ERK activation by assembling signaling complexes comprising Raf-1, MEK, and ERK.[83] The b-arrestin-scaffolded ERK complex accompanies GPCRs to early endosomes, [84] serving the role of dispatching ERK signaling to different subcellular compartments. Importantly, b-arrestins prevent ERK nuclear translocation, favoring ERKs cytosolic functions.[83] A similar effect is observed for Sef, which sequesters activated ERKs at the GC, impeding their nuclear translocation[80] and inhibiting PC12 differentiation.[85] MP1 is a widely expressed scaffold that exclusively binds to the isoforms MEK1 and ERK1.[86] MP1 forms a complex with p14 that targets MP1 and its binding partners to late endosomes. Both MP1 and p14 are essential for EGF-dependent activation of ERK.[79] Interestingly, the positive regulation of ERK signals by MP1 is dependent on its correct placement, as mislocalization to the PM dampens the duration of ERK activation.[79]

Local intervention for the prevention of global malignancy?

The above compilation of data reects how much we have learned since the importance of where for Ras-ERK signaling became recognized, about a decade ago. At the same time, it shows that we have just barely scraped the surface. Even though our understanding of signal compartmentalization is still scant, what we already know is sufcient to draw an important conclusion regarding the application of this knowledge to anti-tumor therapy: the concept of inhibiting specic, localization-dened subsignals to prevent/revert malignant transformation, as an alternative to suppressing the total signal, is valid. Annexin A6 acts as a tumor suppressor in many cell types, by virtue of its ability to recruit p120 GAP specically to non-raft PM microdomains.[44] Transformation of murine broblasts by oncogenes, such as v-Src and Sis, can be forestalled just by inhibiting Ras signals emanating from lipid rafts or disordered membrane.[51] Disrupting Galectin-1 function dislodges H-RasV12 from PM nanoclusters and prevents broblast transformation and PC12 differentiation.[37] These data illustrate that it is not necessary to suppress Ras signaling totally to obtain growth/transformation-suppressive responses, the inhibition of dened site-specic subsignals can be sufcient. The same concept applies to ERKs: blocking ERKs nuclear signaling by PEA15 prevents breast carcinoma cell proliferation and invasion.[91] The same strategy is utilized for the induction of apoptosis, by the pro-apoptotic protein Bik, in lung epithelial cells[92] and by the death-associated

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protein kinase DAPK in cervical carcinoma cells.[93] Likewise, the inhibition of ERKs cytoplasmic component, by preventing ERK dimerization, is sufcient to abrogate broblast transformation and proliferation, and tumor formation in vivo by lung, colorectal, and bladder carcinoma cells.[90] These data indicate that both spatial components of ERKs signals are necessary for tumor development and that interfering with either of them can have remarkable anti-tumor effects. Hitherto, we have learned that Ras-ERK signals can support cellular transformation from different subcellular localizations, but we are still missing a critical piece of information: what localizations in which tumors? Unfortunately, we do not have a clue as to which signaling platform/s is/are critical for driving tumor progression in different cell types. It is very likely that a broad variability will be encountered depending on the cellular context. In spite of this important limitation, we have thus far gathered enough information so as to place in the bulls eye a handful of proteins having potential to become therapeutic targets for inhibiting site-specic Ras signals. Needless to say, that to specically interfere with some local signal we must forget about the main players, Ras-Raf-MEK-ERK, and focus on site-specic ancillary proteins. Galectin-1 could be an attractive candidate as it is involved in cancer in many ways[94] and abrogating its function could disrupt Ras signals coming from H-Ras nanoclusters to prevent cellular transformation.[37] However, Galectin-1 seems to be quite pleiotropic,[95] a drawback that must be considered when anticipating potential toxic effects. The case is not so clear with Galectin-3, as its down-regulation potentiates ERK activation and K-RasV12induced transformation.[36] ERK scaffold proteins are indeed candidates with an enormous potential to become site-specic therapeutic targets. As mentioned before, they recently emerged as important spacerestricted modulators of ERKs signals. Some of them, like KSR1 and MP1, have no other known function but to regulate ERKs, and so, in principle, no pleiotropy-based undesired effects should be expected. Genetic ablation of p14, the scaffold protein through which MP-1 binds to endosomes, resulted in severe effect on the epidermis,[65] which, a priori, raises concerns about the safety of targeting the p14-MP1-ERK complex. It will be important to learn whether MP-1 deletion recapitulates this phenotype. In contrast, disruption of KSR1 yielded grossly normal animals. In these, ERK activation was attenuated and tumor growth signicantly retarded.[96] This could make KSR1 an ideal target for intervention in tumors in which Ras signals derived from lipid rafts play an essential role. Unfortunately, with respect to their structure, most scaffold proteins have turned out to be very difcult to work with, and we are still lacking important structural information on, for example, how MEK and ERK dock onto KSR1. These sites could be two potential hotspots for the design of drugs aimed at disrupting ERKs signal through competitive binding to KSR1. ERKs nuclear and cytoplasmic components are also potential subjects for therapeutic intervention. As mentioned before, sequestering ERKs at the cytoplasm, whereby ERKs nuclear signals are impeded, is sufcient for abrogating growth and/ or potentiating apoptosis.[9193] Recently, a nuclear translocation signal: Ser-Pro-Ser, has been identied within ERKs

insert. These serines are phosphorylated upon stimulation, promoting ERKs translocation to the nucleus.[73] Conceptually, drugs targeted to mask this short sequence, thereby preventing its phosphorylation, could represent a potential strategy to stop ERK nuclear translocation. Blocking ERKs cytoplasmic component through the disruption of ERK dimerization, is sufcient to prevent tumor progression.[90] This demonstrates that even though ERKs nuclear signals are the most efcient for inducing proliferation and transformation,[97] ERKs cytoplasmic component is also necessary for these processes. Importantly, ERKs dimerization interface[88] is probably unique regarding its molecular interactions. Thus, it could provide a highly specic target for drugs aimed at inhibiting ERKs cytoplasmic signals. This venue is being currently explored in our laboratory. Rather than inhibiting a given site-specic subsignal, an alternative strategy is conceivable for tumors that harbor oncogenic Ras: the relocation of mutated Ras to another sublocalization where its signals are less harmful. For example, oncogenic K-Ras, active at the PM where it generates proliferative signals, could be forced to translocate to mitochondria, where it induces apoptosis.[27] Likewise, oncogenic H- or NRas could be redirected to endosomes where ERK activation is less efcient and their transformation potential less pronounced.[31] Unfortunately, while these strategies may be conceptually valid, their technical feasibility, at this moment, is still not clear. In the event that we obtained a drug capable of specically inhibiting some local signal, would it turn out be less toxic than the total signal inhibitors already in use? Of course, the answer to this cannot be anything else but empirical. But, arguably, if some spatially dened signal is critical for proliferation/survival in tumor cells, it should be just as important in normal cells. Or maybe not? It is becoming accepted that tumor cells evolve to become highly dependent on the signals activated by their propelling oncogenic lesions, in a process that has been termed as oncogene addiction.[98] By the same token, should not a tumor cell become addicted to a prevailing site-specic subsignal? What if such subsignal was quantitatively or qualitatively different in tumor cells compared to normal cells? This may very well be the case, for example, nuclear levels of total and phosphorylated ERKs are much higher in renal carcinoma cells than in normal kidney cells.[75] If subsignal addiction does occur, renal carcinoma cells should be much more sensitive to the blockade of ERK nuclear component than its normal counterparts. What about Ras? Are its subsignals different in Rastransformed versus normal cells? Much to our distress, we cannot provide an answer yet. To date, there are no data comparing Ras distribution in tumor and in normal cells, but, making an educated guess, it is likely to be quite different. As mentioned before, H-Ras localization seems to be dependent on its activation state.[17] As such, whereas in normal cells wild-type H-Ras would trafc between raft and non-raft microdomains, in cells harboring the oncogene, H-RasV12 will be locked at non-raft sublocalizations, making these cells more sensitive to therapies directed to specically blocking signals at such compartment. The differences in subcellular distribution between wild-type and activated H/N-Ras may be even greater: Ras-GTP is depalmitoylated much faster than Ras-

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GDP.[99] In addition, some proteins involved in Ras modication, such as DHHC9 palmitoyl transferase, are overexpressed in tumor cells.[100] Thus, these alterations in the machinery that ultimately orchestrates Ras association to different types of sublocalizations may result in gross differences in the subcellular distribution of normal versus oncogenic Ras.

Conclusions
Although still at its infancy, the eld of signal compartmentalization has already provided us with valuable lessons while studying Ras-ERK signaling. We now know that Ras-ERK signals can emanate from distinct subcellular localizations. In some of these places, Ras-ERK signals are distinctively orchestrated by local regulators and processes that, one way or another, make then unique. We have also learned that Ras-ERK signals can support proliferation and survival from several of these localizations. We have also discovered that it is not essential to completely inhibit Ras-ERK signals to block cellular transformation and tumor progression, we can accomplish this goal just by interfering with local subsignals. What could site-specic drugs look like? Of course, at this stage it is highly speculative, but being prophets for a moment, it is likely that traditional kinase inhibitors will not play a role. The fact that Raf is inhibited by some phospholipids offers a promising strategy. Generating drugs that mimic these inhibitory molecules while analyzing the afnity of these lipids for different subcellular locations in normal and tumor cells, could be a valid strategy. Finally, although purely hypothetical at this moment, molecules that compete with ERK or MEK for binding to specic scaffolds, may also be promising candidates. Importantly, it is very likely that what we have learned from Ras-ERK can be, to a large extent, extrapolated to other signaling pathways. These concepts provide us with valuable intellectual venues to begin thinking about how to interfere with local signaling processes to forestall cancer, while waiting for the unveiling of new site-specic regulators that can serve as anti-tumor targets. We are condent that the future shall provide several of these. Acknowledgments n, and R. Seger for We are indebted to R. R. Mattingly, J. Leo their constructive critiques. The laboratory of P.C. is supported by grants BFU2008-01728 from the Spanish Ministry of Education; GROWTHSTOP (LSHC CT-2006-037731) and SIMAP (IST-2004-027265) projects from the EU VI tica de Investigacio n Framework Program and Red Tema ncer (RTICC) (RD06/0020/0105), Spanish Cooperativa en Ca Ministry of Health.

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