This action might not be possible to undo. Are you sure you want to continue?
The Ras-ERK pathway: Understanding site-speciﬁc signaling provides hope of new anti-tumor therapies
´n ˜ez and Piero CrespoÃ Fernando Calvo, Lorena Agudo-Iba
Recent discoveries have suggested the concept that intracellular signals are the sum of multiple, site-speciﬁed 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-speciﬁc 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 ﬁndings 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
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 ﬁgure 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%. Added to this, the experimental evidence substantiating the importance of the Ras-ERK pathway in cancer initiation and progression is
´a de Cantabria (IBBTEC), Consejo Instituto de Biomedicina y Biotecnologı ´ﬁcas (CSIC), IDICAN, Universidad de Superior de Investigaciones Cientı Cantabria, Cantabria, Spain *Corresponding author: Piero Crespo E-mail: firstname.lastname@example.org 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
Bioessays 32: 412–421,ß 2010 WILEY Periodicals, Inc.
and K-Ras4A are further modiﬁed by one or two palmitates. 10] The three Ras isoforms were also detected in endosomes[11. in protocols where ERK phosphorylation is reduced signiﬁcantly.. Further complexity was introduced when it was found that. we review the current knowledge on the compartmentalization of Ras-ERK signals.. GDP-bound. both by academia and industry. Preclinical studies suggest that complete and continuous suppression of ERK activation would be required for maximal therapeutic effects. like the nucleus and the cytoplasm. Ras proteins are no longer thought to be stationary.[17. 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. duration and. within the PM. We now know that Ras is present in various membrane systems where its signals are regulated in distinct manners. The frequency of activating mutations in different human neoplasias is shown. the downstream kinases Raf and MEK remained attractive candidates. K-Ras4B second signal consists of a polybasic region. thanks to the pioneering work of M. there has always been concern that an effective blockade of ERK may just prove too toxic for normal cells. Aiming at components of the Ras-ERK pathway as an antitumor strategy is an old story not devoid of disappointments. Figure 1. considering the central role played by ERK in the regulation of physiological proliferation. 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 efﬁcient while less toxic inhibitors? An opportunity may have arisen following recent discoveries that demonstrate the spatial segregation of Ras-ERK signals. The cysteine in the CAAX box is recognized by farnesyltransferases that catalyze the addition of a farnesyl isoprenoid. mainly by the use of farnesyltransferase inhibitors. fatigue. skin rash. Ras sublocalization appeared to depend on its activation status: inactive. and in disordered membrane domains. it was demonstrated that Ras was also present and active in endomembranes like the endoplasmic reticulum (ER) and the Golgi complex (GC). For stable membrane association Ras requires a second signal: H-Ras. Ras segregated at distinct microdomains: H. Raf. with emphasis on the potential applications to the design of new generations of anti-tumor drugs. ﬁrst to ﬁnd ‘‘drugable’’ targets among its constituents and. while sparing normal ones? Here.[1. More recently..and N-Ras were present in cholesterol-rich domains. such as the one for PD0325901. clinical trials with these have been largely unsuccessful (for a review see ref. including its main components. it is hardly surprising that this signaling cascade has attracted enormous attention for antineoplastic intervention. Calvo et al. where they are inﬂuenced by a broad collection of site-speciﬁc regulators.[9. subsequently. to generate compounds aimed at such signaling intermediaries. 4). Inc. Philips. However. 413 .ß 2010 WILEY Periodicals. clinical trials with several Raf and MEK inhibitors have revealed toxic effects such as diarrhea. etc. 12] and mitochondria[13–15] (for a review see ref. proteolysis. ERK signals unfold in different environments. this has prompted its discontinuation (for extensive reviews see ref. Initially. that exerts a strong electrostatic interaction with membrane phospholipids. which undergoes a series of modiﬁcations including prenylation. translocating to disordered membrane when GTP-loaded. Do these spatially restricted subsignals contribute to the same extent and in the same way to tumorigenesis? Are site-speciﬁc Ras-ERK subsignals similar in normal versus tumor cells? Could we exploit some difference for targeting tumor cells more efﬁciently. This modiﬁcation takes place at the GC. Such a widespread distribution is possible mainly due to the post-translational processing experienced by Ras proteins. substantial efforts were devoted to the inhibition of activated Ras proteins. Thus. Initially. particularly those forming epithelia with a high turn-over. rich in lysine residues. but rather they are known to translocate between Bioessays 32: 412–421. Importantly. which permits a transient association to the ER. visual disturbances. 16). 19). But. 18] Importantly. subsequently. whereas K-Ras was only detected in the latter. Similarly.. In extreme cases. Colossal efforts have been devoted. A simpliﬁed representation of the Ras-ERK pathway. and carboxyl methylation (for a review see ref. also known as lipid rafts. 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. Review essays overwhelming: activated mutants of Ras. Ras was thought to act exclusively at the plasma membrane (PM). and MEK have been shown to induce malignant transformation in many cell types. With Ras out of the limelight.  and ). Prospects & Overviews F. although far from completely and continuously. 2] and sophisticated animal models have endorsed the importance of this pathway for tumorigenesis in vivo. But. directly affects therapeutic efﬁcacy. H-Ras associated predominantly to lipid rafts. N-Ras. These are synthesized as hydrophilic proteins terminating in a CAAX motif.
. Inc. by forming a complex therein with p120 GAP. and recycled back to the GC probably escorted by some chaperone that insulates the hydrophobic isoprenyl moiety. Following stimulation. The presence of Ras in endosomes. to the GC. facilitating its translocation to the GC and to early endosomes. Active H-Ras is internalized together with EGF receptors after stimulation.[11. These ancillary proteins are critical for the formation of Ras activation and activities: When site matters Not unexpectedly. GAPs. via retrograde transport. guanine nucleotide exchange factors (GEFs) were thought to activate Ras exclusively at the PM. 26] Non-palmitoylated K-Ras4B also trafﬁcs back and forth from the PM. thereby redirecting signaling to K-Ras nanoclusters. although by other mechanisms: phosphorylation of K-Ras4B by PKC promotes its release from the PM and its transfer to mitochondria. inducing the formation of activated H-Ras/Galectin-1 complexes. H. A consequence of the site-speciﬁc activity of GEFs. Later. Galectin-1 not only behaves as a key scaffold for the formation of H-Ras nanoclusters. N.and H-Ras establish stable interactions with the endosomal compartment. Review essays Figure 2. to their ﬁnal destinations at the PM. Galectin-1 is recruited to the PM in response to H-Ras activation. Calvo et al. by ubiquitination. For example.ß 2010 WILEY Periodicals. but not of K-Ras. is signiﬁcant. Calmodulin binding to K-Ras4B also dislodges it from the PM. The way Ras is activated is also inﬂuenced by. K-Ras does not. Once there.and H-Ras undergo a palmitoylation/depalmitoylation cycle. Another ancillary protein. Site-speciﬁc effects have also been observed for the deactivation of Ras by GTPase activating proteins (GAPs). whereby palmitoylated forms at the PM are released following depalmitoylation. cellular compartments. Interestingly.. 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.and H-Ras.and N-Ras are palmitoylated at the GC by acyl-transferases of the DHCC9/GCP16 families. Galectin-3 that inhibits Ras-GRP4 to divert incoming signals away from H. by returning depalmitoylated H-Ras to the GC. Ras association to endosomes exhibits isoform speciﬁcity: whereas N. Ras proteins partition into microdomains referred to as ‘‘nanoclusters.’’ Formation of these nanostructures requires the interaction with Galectins. palmitoylation is rapidly reversible.[25. From there. At the PM. Originally.F. it was found that Ras was also switched-on at different endomembranes and by distinct GEFs. and trafﬁc. Galectin-3 functions in a similar way. for example. promotes Ras inactivation in non-raft PM microdomains and in PM-derived endosomes. these become enriched in cholesterol-independent microdomains. but speciﬁcally for K-Ras. and other regulatory proteins is that such compartmentalization 414 Bioessays 32: 412–421. Ras proteins are repalmitoylated and sent back to the PM by vesicular transport (Fig. Annexin A6. There. 41] whereas SOS and Ras-GRF GEFs function at the ER. they are depalmitoylated. in lymphocytes. it promotes Ras activation at the GC through Ras-GRP1 and triggers PM Ras inactivation by the calcium-responsive GAP CAPRI. Ras acylation cycle. via vesicular transport. 30. 31] These differences have been attributed to the modiﬁcation of N. Whereas calcium stimulates Ras activation at the PM using Ras-GRF in epithelial cells. they are transported.and N-Ras nanoclusters. and in the endocytic route in general. it also functions as a molecular chaperone that contributes to H-Ras trafﬁcking. mainly by APT-1 acyl-thioesterase. Whereas farnesylation is fairly stable. nanoclusters: antagonizing Galectin-1 results in the displacement of H-Ras from the PM and in the inhibition of Ras biological activity. Ras is activated by Ras-GRP GEFs at the GC. 2).[40.. Prospects & Overviews .
Ultimately. could also be dependent on the membrane composition. PI3K. H-Ras restricted to the GC. each at its own territory. at two different sites. which generates a potent anti-apoptotic signal. some Ras biological effects have been assigned to speciﬁc localizations. and Ral-GDS. This notion also seems to hold at the ‘‘nano’’ scale: EGF stimulates Raf recruitment to K-Ras but not to H-Ras nanoclusters. Ras activation at the PM is fast and transient. In its place. can yield two diametrically opposed biological outcomes. probably due to differences in the GC tethers utilized. 3). at bulk membrane it behaved similarly. In agreement with Raf/ERK activation proﬁles. In agreement. 4). Ras trafﬁcking can also impact on effector utilization. an example of how Ras-ERK signaling can be regulated by conﬁnement to a speciﬁc compartment. proliferation or death. K-Ras. PM versus mitochondria. It is important to realize that Ras localization is not the only source of spatial variability. 38] Importantly. probably due to experimental details. leads to enhanced recruitment of effectors and greater signal output. intensity. linear Ras pathway. For example. whereas Galectin-3 attenuates ERK activation without affecting PI3K activity. each with its own regulators (Fig. transient activation of the Ras-ERK pathway induces proliferation. which may be related to the potentiation of cellular transformation by Galectin-1. the concept of different localizations conferring variability in Ras effector usage has been ﬁrmly established. since conﬁning the GTPase to lipid rafts severely inhibits sustained ERK activation and prevents PC12 differentiation. Most of what we know about Ras compartmentalized functions.. only ER-tethered H-RasV12 could sustain survival in response to growth factor starvation. formation of Ras nanoclusters transforms analog signal input into digital output. In addition. we have learned by the use of artiﬁcially tethered constructs. Such an effect must entail Ras trafﬁcking. In Drosophila. whereas at endomembranes[45. 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. like phosphatidylethanolamine and phosphatidylinositol. Even though some of these results may differ. disordered membrane and lipid rafts. RKTG. these data should be sufﬁcient for abandoning the classical model of one. It has long been recognized that in PC12 cells. this has a lot to do with the abundance and availability of effector molecules at different sites. we should envision several of these pathways. 415 . Ras sublocalization markedly inﬂuences which effector pathways are activated and how intensively. Weaker antigens that induce positive selection generate a milder Ras-ERK signal at the GC. ERKs lose their afﬁnity for these partners and undergo a rapid relocalization to The environment sets the response The aforementioned differences in timing. Calvo et al. a seven transmembrane protein localized at the GC. Prospects & Overviews F. Effector usage can be inﬂuenced by site-speciﬁc regulatory proteins. Ras effectively supported proliferation and cellular transformation of murine ﬁbroblasts from the ER. PI3K.[37.ß 2010 WILEY Periodicals. Inc. Similarly. 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. is particularly enriched at the ER. Probably. Ubiquitination of H. as a consequence of their interaction with several types of cytoplasmic anchors. Review essays Some space for ERKs Even though it may just be the tip of the iceberg. Some of these. By the use of artiﬁcially tethered constructs. 46] and at endosomes is slow and sustained.. a threshold of Ras ubiquitination is required to prevent excess ERK signaling. both outcomes requiring Ras-ERK activation. also suggesting that the GC platform negatively regulates Ras-ERK signals.. This observation demonstrates that the same actor. ERKs are found in the cytoplasm. was also unable to transform. in thymocytes. Negative selection results from a strong PM Ras-ERK activation. Under resting conditions. Once phosphorylated. where reduced availability of Raf results in diminished ERK activation. inhibit their kinase activity. Thus. and intermediaries that deﬁne each site-speciﬁc Ras subsignal Bioessays 32: 412–421. and JNK. 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 efﬁciently activated ERK. N-Ras. ERK activation was also observed when H-RasV12 was tethered to the GC. Seminal studies by Mark Philips showed that H-RasV12 conﬁned to the ER. as exempliﬁed by Galectins: Galectin-1 diverts H-Ras signals to Raf-1 at the expense of PI3K. inhibiting ERK activation and PC12 differentiation. where it induces apoptosis. positive versus negative selection leads to proliferation and apoptosis respectively. results in a remarkable variability in Ras signal outputs. 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. both B-Raf and C-Raf interact with membrane phospholipids. unproductively sequestered Raf into the GC. profusely activated Ral-GDS but not ERK. although in both studies transformation correlates with ERK activation. activated ERK. a phosphatidylethanolamine-binding Raf inhibitor. implying that this endomembrane could be an important site for the regulation of survival. although with lower PI3K activation. the presence of regulatory proteins like RKIP. Such a notion does much to explain the diversity in outputs that Ras signals can achieve. Ras tethered to the GC by the E1 ABV protein. Interestingly. whereas prolonged activation causes differentiation.and N-Ras promotes their association to endosomes. by stabilizing H-Ras nanoclusters. ultimately shape the resulting biological outputs. We now know that ERKs are strictly regulated by multiple elements that modulate their localization and site-speciﬁc activities (Fig. Galectin-1. in murine ﬁbroblasts. but not from GC. Such is the case for ERK 1 and 2 (ERKs) MAP kinases. Furthermore. Other components of the underlying cascades contribute in their own way. A discrepancy exists with respect to Ras transforming potential at the GC.
Calvo et al. Ras compartmentalized functions and site-speciﬁc regulators. resulting in apoptosis.. . cal.. Inc. when presence is dependent on modiﬁcations: ub. calmodulin binding. Prospects & Overviews .F. Review essays Figure 3. and DAPK. The isoforms present stably at each subcellular compartment are shown in blue. Ancillary proteins speciﬁcally acting at particular sites are also depicted. ubiquitination. phosphorylation.ß 2010 WILEY Periodicals. ERKs nuclear translocation can be prevented by proteins like PEA15. P. 416 Bioessays 32: 412–421. Scaffold proteins acting at speciﬁc sublocalizations are shown. Bik. In purple. Figure 4. Compartmentalized regulation of ERKs. ERKs functions at the cytoplasm are undertaken mainly as dimers whereas it translocates to the nucleus as monomers.
 For efﬁcient nuclear translocation. endosomal trafﬁc. However. are non-nuclear proteins. insulating a MAPK module from undesired interferences. dimerization was proposed to be important for ERK nuclear import. As such. ERKs are known to dimerize upon phosphorylation. chromatin remodeling. Even though our understanding of signal compartmentalization is still scant. Importantly. Review essays A place for a scaffold In addition to the kinase tiers. ERK functions within the nucleus may also be compartmentalized further: ERKs interact with lamin A at the nuclear envelope to promote rapid. The main function of scaffolds is to bring together the members of the signaling cascade.. ERKs extranuclear component is just as important. In the nucleus. The same concept applies to ERKs: blocking ERKs nuclear signaling by PEA15 prevents breast carcinoma cell proliferation and invasion. KSR1 is not the only scaffold utilized by lipid raft-generated signals. Transformation of murine ﬁbroblasts by oncogenes. with such substrate speciﬁcity being governed by the participation of distinct scaffold proteins. These data illustrate that it is not necessary to suppress Ras signaling totally to obtain growth/transformation-suppressive responses. the nucleus. it shows that we have just barely scraped the surface. MP1 is a widely expressed scaffold that exclusively binds to the isoforms MEK1 and ERK1. Sef at the GC. and spatial speciﬁcity of its signals. as an alternative to suppressing the total signal. They enhance ERK activation by assembling signaling complexes comprising Raf-1. MP1 forms a complex with p14 that targets MP1 and its binding partners to late endosomes. but in this case the mediating scaffold is KSR1. MEK. ERKs perform essential functions that regulate transcription. prevent ERKs nuclear translocation and in doing so. favoring ERKs cytosolic functions. promote ERK cytoplasmic activities. impeding their nuclear translocation and inhibiting PC12 differentiation. In addition. scaffolds and substrates to help explain how scaffolds achieve spatial selectivity for ERKs signals. in most cases. and anti-apoptotic signaling are dependent on ERK extranuclear activity. and miRNA synthesis[60. and ERK. At the same time. by virtue of its ability to recruit p120 GAP speciﬁcally to non-raft PM microdomains. scaffolds play important roles in the spatial selectivity of ERK signals: KSR1 acts preferentially upon ERK signals emanating from PM cholesterol-rich domains. In contrast. such as v-Src and Sis. intensity. Annexin A6 acts as a tumor suppressor in many cell types. since the phosphorylation of another ERK substrate. We have recently demonstrated that scaffold proteins serve as dimerization platforms in which ERK dimers are assembled. 417 . mitogen-dependent AP-1 activation. adhesion. the ERK cascade includes other proteins that regulate the amplitude. ERKs require direct interaction with the nuclear pore complex. The same strategy is utilized for the induction of apoptosis. Interestingly. Interestingly.. At the same time. a nuclear translocation signal within their ‘‘insert’’ domain and the participation of nuclear shuttles like Importin-7 or Mxi2. Initially. The microenvironment from which Ras signals emanate determines which substrates will be preferentially phosphorylated by activated ERKs. is valid.. the inhibition of deﬁned site-speciﬁc subsignals can be sufﬁcient. cPLA2 is also activated by ERKs in response to Ras signals coming from lipid rafts. by releasing cFos from its inhibitory interaction with lamin A. MP-1 regulates ERK in endosomes. Prospects & Overviews F. The b-arrestin-scaffolded ERK complex accompanies GPCRs to early endosomes. forming a complex which helps in optimizing the signal.ß 2010 WILEY Periodicals. but it was later shown that ERKs nuclear inﬂux occurs mainly in monomeric form. Local intervention for the prevention of global malignancy? The above compilation of data reﬂects how much we have learned since the importance of ‘‘where’’ for Ras-ERK signaling became recognized. It fact. b-Arrestins are abundant in clathrin-coated pits. like the promotion of proliferation. such as the modulation of integrin receptors. It has been estimated that half of the ERKs content remains in the cytoplasm after stimulation and processes such as the formation of cell-matrix contacts. is mediated by IQGAP. nearly half of the $180 proteins thus far identiﬁed as ERKs substrates. The nucleo-cytoplasmic shuttling of ERKs is exquisitely regulated. leaving dimers orphans of a function. like PEA15. the positive regulation of ERK signals by MP1 is dependent on its correct placement. activation of ERKs nuclear substrates does not require the participation of scaffolds. about a decade ago. which sequesters activated ERKs at the GC.  serving the role of dispatching ERK signaling to different subcellular compartments. A similar effect is observed for Sef. Calvo et al. can be forestalled just by inhibiting Ras signals emanating from lipid rafts or disordered membrane. and is chieﬂy undertaken by ERK monomers. Another space-related regulatory mechanism that has been disclosed recently concerns ERK dimerization. by the pro-apoptotic protein Bik. dimerization and scaffolding appear to be essential processes for ERKs cytoplasmic but not nuclear signaling. Recent ﬁndings from our laboratory have put together space. b-arrestins prevent ERK nuclear translocation. what we already know is sufﬁcient to draw an important conclusion regarding the application of this knowledge to anti-tumor therapy: the concept of inhibiting speciﬁc. Golgi fragmentation. EGFr. localization-deﬁned subsignals to prevent/revert malignant transformation. These scaffold-dimer complexes are essential for the ensuing interaction of ERKs with their cognate cytoplasmic substrates. and Paxillin at focal adhesions. Speciﬁcally. while interfering with ERKs nuclear functions. in lung epithelial cells and by the death-associated Bioessays 32: 412–421. as mislocalization to the PM dampens the duration of ERK activation. Some proteins. Scaffold proteins serve all of these purposes. Both MP1 and p14 are essential for EGF-dependent activation of ERK. Inc. ERKs activated by Ras at the ER utilize Sef-1 to activate cPLA2. Disrupting Galectin-1 function dislodges H-RasV12 from PM nanoclusters and prevents ﬁbroblast transformation and PC12 differentiation. where they phosphorylate multiple nuclear proteins. they exclude other related components that operate in parallel cascades. DNA replication. 61] among others.
[91–93] Recently. renal carcinoma cells should be much more sensitive to the blockade of ERK nuclear component than its normal counterparts. would it turn out be less toxic than the ‘‘total’’ signal inhibitors already in use? Of course. where it induces apoptosis. resulted in severe effect on the epidermis. For example. Galectin-1 seems to be quite pleiotropic. we do not have a clue as to which signaling platform/s is/are critical for driving tumor progression in different cell types.. drugs targeted to mask this short sequence. and bladder carcinoma cells. oncogenic H. in cells harboring the oncogene. at this moment. a priori. could be forced to translocate to mitochondria. is sufﬁcient for abrogating growth and/ or potentiating apoptosis. a nuclear translocation signal: Ser-Pro-Ser. how MEK and ERK dock onto KSR1. oncogenic K-Ras. Importantly.or NRas could be redirected to endosomes where ERK activation is less efﬁcient and their transformation potential less pronounced. What about Ras? Are its subsignals different in Rastransformed versus normal cells? Much to our distress. for example. Prospects & Overviews . and we are still lacking important structural information on.ß 2010 WILEY Periodicals. in a process that has been termed as ‘‘oncogene addiction. As mentioned before. in principle. is still not clear. active at the PM where it generates proliferative signals. is sufﬁcient to prevent tumor progression. ERK activation was attenuated and tumor growth signiﬁcantly retarded. but we are still missing a critical piece of information: what localizations in which tumors? Unfortunately. colorectal. It will be important to learn whether MP-1 deletion recapitulates this phenotype. if some spatially deﬁned signal is critical for proliferation/survival in tumor cells. Calvo et al. H-Ras localization seems to be dependent on its activation state. Inc. thereby preventing its phosphorylation. Unfortunately. Hitherto. These sites could be two potential hotspots for the design of drugs aimed at disrupting ERKs signal through competitive binding to KSR1. Or maybe not? It is becoming accepted that tumor cells evolve to become highly dependent on the signals activated by their propelling oncogenic lesions. whereby ERKs nuclear signals are impeded. Some of them. could represent a potential strategy to stop ERK nuclear translocation. To date. is sufﬁcient to abrogate ﬁbroblast transformation and proliferation. This demonstrates that even though ERKs nuclear signals are the most efﬁcient for inducing proliferation and transformation. Likewise. we cannot provide an answer yet. promoting ERKs translocation to the nucleus. their technical feasibility. should not a tumor cell become addicted to a prevailing site-speciﬁc subsignal? What if such subsignal was quantitatively or qualitatively different in tumor cells compared to normal cells? This may very well be the case. most scaffold proteins have turned out to be very difﬁcult to work with. Unfortunately. as its down-regulation potentiates ERK activation and K-RasV12induced transformation. . nuclear levels of total and phosphorylated ERKs are much higher in renal carcinoma cells than in normal kidney cells. In contrast. which. and tumor formation in vivo by lung. As such.’’ These serines are phosphorylated upon stimulation. H-RasV12 will be locked at non-raft sublocalizations. Blocking ERKs cytoplasmic component through the disruption of ERK dimerization. Thus. making an educated guess. In the event that we obtained a drug capable of speciﬁcally inhibiting some local signal. a drawback that must be considered when anticipating potential toxic effects. Genetic ablation of p14. ERKs dimerization interface is probably unique regarding its molecular interactions. but. Likewise. disruption of KSR1 yielded grossly normal animals.’’ By the same token. by preventing ERK dimerization. we have learned that Ras-ERK signals can support cellular transformation from different subcellular localizations. Conceptually. arguably. for example. raises concerns about the safety of targeting the p14-MP1-ERK complex. protein kinase DAPK in cervical carcinoma cells. 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.. they recently emerged as important spacerestricted modulators of ERKs signals. the answer to this cannot be anything else but empirical. If ‘‘subsignal addiction’’ does occur. ERKs cytoplasmic component is also necessary for these processes. and focus on site-speciﬁc ancillary proteins. and so. the inhibition of ERKs cytoplasmic component. However. 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. the scaffold protein through which MP-1 binds to endosomes. ERKs nuclear and cytoplasmic components are also potential subjects for therapeutic intervention. But. Ras-Raf-MEK-ERK. we have thus far gathered enough information so as to place ‘‘in the bull’s eye’’ a handful of proteins having potential to become therapeutic targets for inhibiting site-speciﬁc Ras signals. ERK scaffold proteins are indeed candidates with an enormous potential to become site-speciﬁc therapeutic targets. Rather than inhibiting a given site-speciﬁc subsignal. Needless to say. there are no data comparing Ras distribution in tumor and in normal cells. that to speciﬁcally interfere with some local signal we must forget about the main players. This could make KSR1 an ideal target for intervention in tumors in which Ras signals derived from lipid rafts play an essential role. no pleiotropy-based undesired effects should be expected. As mentioned before. 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- Review essays 418 Bioessays 32: 412–421. This venue is being currently explored in our laboratory. has been identiﬁed within ERKs ‘‘insert. it should be just as important in normal cells. It is very likely that a broad variability will be encountered depending on the cellular context. have no other known function but to regulate ERKs. Galectin-1 could be an attractive candidate as it is involved in cancer in many ways and abrogating its function could disrupt Ras signals coming from H-Ras nanoclusters to prevent cellular transformation.F. with respect to their structure. while these strategies may be conceptually valid. making these cells more sensitive to therapies directed to speciﬁcally blocking signals at such compartment. it is likely to be quite different. whereas in normal cells wild-type H-Ras would trafﬁc between raft and non-raft microdomains. like KSR1 and MP1. In spite of this important limitation. it could provide a highly speciﬁc target for drugs aimed at inhibiting ERKs cytoplasmic signals. The case is not so clear with Galectin-3. sequestering ERKs at the cytoplasm. In these. As mentioned before.
2. Silvius JR. Spanish Cooperativa en Ca Ministry of Health. et al. Drake KR. We now know that Ras-ERK signals can emanate from distinct subcellular localizations. 1999. Chiu VK. Importantly. Willingham MC. Endomembrane trafﬁcking of Ras: the CAAX motif targets proteins to the ER and Golgi. Rocks O. Mol Cell Biol 22: 5128– 40. Rogers C. Peyker A. 23.. Conclusions Although still at its infancy. 25. some proteins involved in Ras modiﬁcation. We are conﬁdent that the future shall provide several of these. Wolfman JC. Nat Rev Cancer 4: 937–47. Childs JE. Leo their constructive critiques. Hamilton AD. Zhu K. Pastan I. 18. Montagut C. Philips M. 7. et al. 26. to a large extent. Identiﬁcation of a Ras palmitoyltransferase in Saccharomyces cerevisiae. Bar-Sagi D. 6. References 1. Roy S. An acylation cycle regulates localization and activity of palmitoylated Ras isoforms.C. Curr Opin Investig Drugs 4: 1428– 35. is supported by grants BFU2008-01728 from the Spanish Ministry of Education. We have also discovered that it is not essential to completely inhibit Ras-ERK signals to block cellular transformation and tumor progression. 419 . the ﬁeld of signal compartmentalization has already provided us with valuable lessons while studying Ras-ERK signaling. 1999. et al. J Biol Chem 277: 41268–73. Compartmentalized signalling of Ras. PKC regulates a farnesyl-electrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis. 2009. Hancock JF. 29. Nat Cell Biol 3: 368–75. Biochem J 389: 1–11. Bivona TG. 2009. Magee AI. Cancer Lett 283: 125–34. Weinberg RA. 14. Wong KK. while waiting for the unveiling of new site-speciﬁc regulators that can serve as anti-tumor targets. 19. 27. Oncogene 26: 3291–310. Hancock JF. one way or another. Mechanisms of Ras protein targeting in mammalian cells. 8. et al. Fehrenbacher N. Shih TY. at this stage it is highly speculative. Matallanas D. 17. 2009. 11. Kirschmeier P. The laboratory of P. Yan J. Mattingly. J Membr Biol 190: 83–92. Bcl-2 differentially targets K-. Curr Opin Genet Dev 19: 4–11. Sorkin A. Ras proteins are polyisoprenylated but only some are palmytoylated. Prior IA. it is very likely that what we have learned from Ras-ERK can be. 2009. 2002. 24. Review essays Bioessays 32: 412–421. 5. Generating drugs that mimic these inhibitory molecules while analyzing the afﬁnity of these lipids for different subcellular locations in normal and tumor cells. Martinez AC.. Characterization of Haras. et al. et al. J Biol Chem 275: 19315–23. 1980. Wolfman A. 1997. Differences in the inhibitory speciﬁcities of H-Ras. Settleman J. 2006. Acknowledgments ´ n. J Cell Biol 170: 261–72. 2005. Gomez GA. Sebolt-Leopold JS. Der CJ. it is likely that ‘‘traditional’’ kinase inhibitors will not play a role. Mol Biol Cell 13: 1522–35. et al. What could site-speciﬁc drugs look like? Of course. J Cell Biol 170: 429–41. Ras-ERK signals are distinctively orchestrated by local regulators and processes that. Fivaz M. 1990.ß 2010 WILEY Periodicals. 10. Choy E. Hancock JF. Berciano MT. 12. Calvo et al. Lobo S. but being prophets for a moment. we can accomplish this goal just by interfering with local subsignals. Quatela SE. Linder ME. and H-Ras to mitochondria in IL-2 supplemented or deprived cells: implications in prevention of apoptosis. 1989. Parton RG. J. 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). molecules that compete with ERK or MEK for binding to speciﬁc scaffolds. N-. could be a valid strategy. H-Ras dynamically interacts with recycling endosomes in CHO-K1 cells: involvement of Rab5 and Rab11 in the trafﬁcking of H-Ras to this pericentriolar endocytic compartment. Tuveson DA. Reversible intracellular translocation of KRas but not HRas in hippocampal neurons regulated by Ca2þ/calmodulin. Ras plasma membrane signalling platforms. Wyse B. N-ras. Thus. Kahms M. 2005. 2004. 1987. 4. Herrera R. 2007. 2005. Karnoub AE. 15. Harding A. GDP. Farnesyltransferase inhibitors as anticancer agents: current status. McKay IA. 20. Marshall CJ. Mol Cell 21: 481–93. 2005. Hancock JF. Perez-Sala D. Recent Pat Anticancer Drug Discov 4: 28–35. 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. Zhang FL. 2002. Depalmitoylated Ras trafﬁcs to and from the Golgi complex via a nonvesicular pathway. We have also learned that Ras-ERK signals can support proliferation and survival from several of these localizations. Silletti J. Ki-Ras4A. Biochem Soc Trans 33: 657–61. Prospects & Overviews F. Recent developments in anti-cancer agents targeting the Ras/Raf/ MEK/ERK pathway. et al. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Paterson H. 2000. R. Goodwin JS. Bodemann BO. such as DHHC9 palmitoyl transferase. Mol Oncol 3: 297–307. J Biol Chem 278: 4572–81. et al. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Endogenous c-N-Ras provides a steady-state anti-apoptotic signal. and R. 2002. Rebollo A.. Philips MR. 2005. In some of these places. Targeting the RAF-MEK-ERK pathway in cancer therapy. J Biol Chem 272: 10232–9. Sebti SM. 2002. 2001. make then unique. Cell 57: 1167–77. H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis. are overexpressed in tumor cells. Greentree WK. Roberts PJ. Gutierrez L. 9. Ras/MAPK signaling from endomembranes. Seger for We are indebted to R. Carr D. and Ki-Ras4B as in vitro substrates for farnesyl protein transferase and geranylgeranyl protein transferase type I. Cell 19: 1005–14. 2003. 22. Daniotti JL. Jiang X. 3. 16. Cell 63: 133–9. Cell 98: 69–80. et al. Arozarena I. These concepts provide us with valuable intellectual venues to begin thinking about how to interfere with local signaling processes to forestall cancer. 21. Coordinated trafﬁc of Grb2 and Ras during epidermal growth factor receptor endocytosis visualized in living cells. Oncogene 18: 4930–9. may also be promising candidates. Nat Rev Mol Cell Biol 9: 517–31. Science 307: 1746–52. The fact that Raf is inhibited by some phospholipids offers a promising strategy. 2008. Targeting the mitogen-activated protein kinase cascade to treat cancer. Magee AI. Finally. Meyer T. Localization of the src gene product of the Harvey strain of MSV to plasma membrane of transformed cells by electron microscopic immunocytochemistry. Karreth FA. Inc. K-Ras and N-Ras (N17) dominant negative mutants are related to their membrane microlocalization. 2005. 2003. Ras oncogenes: split personalities. GTP-dependent segregation of H-ras from lipid rafts is required for biological activity. Modelling oncogenic Ras/Raf signalling in the mouse. J Biol Chem 280: 34997–5010. et al. although purely hypothetical at this moment. EMBO J 6: 3353–7. extrapolated to other signaling pathways. In addition. Dynamic fatty acylation of p21N-ras. 13. 28.
Death effector domain protein PEA-15 potentiates Ras activation of extracellular signal receptor-activated kinase by an adhesion-independent mechanism. Biochim Biophys Acta 1783: 985–93. ERK1/2. 2007. Bioessays 28: 1211–20. Plowman SJ. Frame MC. Direct visualization of Ras proteins in spatially distinct cell surface microdomains. 2009. Seger R. Nunez F.. Clyde-Smith J. 2006. Nature 424: 694–8. et al. Curr Biol 4: 694–701. Oncogene 27: 2754–62. Wilsbacher JL. 2006. et al. Mol Cell 21: 679–87. Cell Signal 20: 1–9. et al. et al. Compartment-speciﬁc feedback loop and regulated trafﬁcking can result in sustained activation of Ras at the Golgi. 2004. 43. J Biol Chem 276: 23341–8. Chin ML. Rotblat B. Arozarena I. Spatial regulation of Raf kinase signaling by RKTG. 2001. Bunch TA. Scotto-Lavino E. Spatiotemporal organization of Ras signaling: rasosomes and the galectin switch. 74. 66. 45. 50. Ye X. Nat Cell Biol 9: 905–14. J Biol Chem 278: 33465–73. Galectin-3 regulates RasGRP4-mediated activation of N-Ras and H-Ras. J Biol Chem 277: 272–8. Hammond DE. Sanchez-Perez I. 47. et al. Wunderlich W. Transcriptomal proﬁling of site-speciﬁc Ras signals. 35. et al. et al. . Differential modiﬁcation of Ras proteins by ubiquitination. 34. Plowman SJ. 2003. Klysik J. Cell 139: 112–22. et al. Ras regulates assembly of mitogenic signalling complexes through the effector protein IMP. et al. 42. Raf-1 and phosphoinositide 3-kinase. et al. Taub N. 2002. Ashery U. Muncke C. 2007. 58. 2008.ß 2010 WILEY Periodicals. Kortum RL. Dev Cell 1: 239–50. Belanis L. J Biol Chem 277: 24090–2102. Parton RG. 2004. 63. 2006. 44. 2003. et al. 38. Mol Biol Cell 16: 3552–61. J Cell Biol 183: 653–66. Levy R. et al. et al. 73. Bivona TG. Ebner HL. Proc Natl Acad Sci USA 92: 8881–5. Elad-Sfadia G. Haklai R. Distinct utilization of effectors and biological outcomes resulting from sitespeciﬁc Ras activation: Ras functions in lipid rafts and Golgi complex are dispensable for proliferation and transformation. Derivatives of activated H-ras lacking C-terminal lipid modiﬁcations retain transforming ability if targeted to the correct subcellular location. et al. Jura N. Ras signaling on the endoplasmic reticulum and the Golgi. Guanine nucleotide exchange factors: activators of the Ras superfamily of proteins. Fauquet M. 2001. 33. 80. Seger R. et al. 2000. 2007. et al. Bioessays 17: 395–404. J Biol Chem 277: 37169–75. Dhanasekaran DN. et al. Thymic selection threshold deﬁned by compartmentalization of Ras/MAPK signalling. 2005. 2007. et al. Yazicioglu MN. 2009. Whitehurst AW. 46. 76. et al. Proc Natl Acad Sci USA 104: 14348–53. J Biol Chem 279: 32813–23. 52. Gill J. 40. Cancer Res 64: 3112–8. Shalom-Feuerstein R. J Cell Biol 175: 861–8. Krishnamoorthy S. Cobb MH. et al. Quilliam LA. Zugaza JL. Mol Biol Cell 19: 1404–14. ERK2 enters the nucleus by a carrier-independent mechanism. Reszka AA. 2006. et al. 2007. Chuderland D. Yoon S. et al. and lipid second messengers: preferential binding of Raf to artiﬁcial lipid rafts. Inc. and c-Fos at the nuclear envelope. James BP. Glading A. Prior IA. 1994. Kusakabe M. Signaling via mitogen-activated protein kinase kinase (MEK1) is required for Golgi fragmentation during mitosis. Niv H. Mitogen-activated protein kinases pathways. Electrostatic interactions positively regulate K-Ras nanocluster formation and function. et al. 67. Makovski V. 30. 70. Inder K. 2008. Huff SY. 2004. et al. Galectin-1 augments Ras activation and diverts Ras signals to Raf-1 at the expense of phosphoinositide 3-kinase. Clague MJ. Sedivy JM. 77. Acharya U. Feng L. 2004. Seedorf K. Mol Biol Cell 18: 4190–9. Localization of the MP1-MAPK scaffold complex to endosomes is mediated by p14 and required for signal transduction. J Cell Sci 122: 1461–70. 53. 79. Glading A. 1998. Paroo Z. Taub N. Hughes PE. 1997. 61. 55. Keyse SM. 2003. Impairment of ubiquitylation by mutation in Drosophila E1 promotes both cell-autonomous and noncell-autonomous Ras-ERK activation in vivo. 64. J Biol Chem 279: 34922–30. 31. Hart KC. 2002. Plasma membrane nanoswitches generate high-ﬁdelity Ras signal transduction. Navarro-Puche A. et al. et al. 69. Goodall A. et al. Review essays 420 Bioessays 32: 412–421. Teixeiro E. the central nervous system and reproduction. Activation of H-Ras in the endoplasmic reticulum by the RasGRF family guanine nucleotide exchange factors. 1995. Mol Biol Cell 18: 4698– 710. Calvo et al. 62. Galectin-1 is a novel structural component and a major regulator of h-ras nanoclusters. Teis D. J Cell Biol 160: 165–70. Cell Signal 19: 2264–76. Eungdamrong NJ. Casar B. Chiu VK. Membrane proximal ERK signaling is required for M-calpain activation downstream of epidermal growth factor receptor signaling. et al. Balan E. You Y. 75. 2008. Xie X. Identiﬁcation and characterization of a general nuclear translocation signal in signaling proteins. Hach A. 49. Oncogene 26: 3185–202. 71. 57. p14-MP1-MEK1 signaling regulates endosomal trafﬁc and cellular proliferation during tissue homeostasis. Sef is a spatial regulator for Ras/MAP kinase signaling. Phosphorylation of phosphoprotein enriched in astrocytes (PEA-15) regulates extracellular signal-regulated kinase-dependent transcription and cell proliferation. Daniels MA. 2008. 2002. Ramos JW. Association of mitogenactivated protein kinase with the microtubule cytoskeleton. Agudo-Ibanez L. EMBO J 26: 635–46. Mol Cell Biol 26: 100–16. Kashef K. Chou FL. Galectin-3 augments KRas activation and triggers a Ras signal that attenuates ERK but not phosphoinositide 3-kinase activity. Nat Cell Biol 4: 343–50. Ramos JW. Active ERK/MAP kinase is targeted to newly forming cell-matrix adhesions by integrin engagement and v-Src. 54. 60. 2008. 2007. et al. Lee CM. Galectin-1(L11A) predicted from a computed galectin-1 farnesyl-binding pocket selectively inhibits RasGTP. Andre S.and C-Raf with cholesterol. Perez de Castro I. Mol Cell Biol 24: 1516–30. Cell Mol Neurobiol 26: 471–95. Caloca MJ. Diltz CD. Villar AV. Ajenjo N. Galectin-1 binds oncogenic H-Ras to mediate Ras membrane anchorage and cell transformation. 56. Tian T. et al. Grewal T. 2008. 2006. 2002. 2002. 36. Donoghue DJ. et al. 51. Mxi2 promotes stimulus-independent ERK nuclear translocation. EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor. Prospects & Overviews . Subcellular localization determines the protective effects of activated ERK2 against distinct apoptogenic stimuli in myeloid leukemia cells. Iyengar R. 2007. Dev Cell 7: 33–44. Chen S. et al. Khosravi-Far R. Casar B. Formstecher E. Traverse S. Hamm H. Ariotti N. 2002. EMBO J 19: 2911–23. Cell 92: 183–92. Renshaw MW. Hekman M. The linker domain of the Ha-Ras hypervariable region regulates interactions with exchange factors. Konson A. 68. Robinson MJ. Yamamoto T. Late endosomal trafﬁc of the epidermal growth factor receptor ensures spatial and temporal ﬁdelity of mitogen-activated protein kinase signaling. Matheny SA. Oncogene 20: 7486–93. Ahearn IM. et al. Horvath EA. Oncogene 14: 945–53. 59. 72.. 2006. Elad-Sfadia G. Seger R. 39. 65. Mol Cell 31: 850–861. et al. Fast regulation of AP-1 activity through interaction of lamin A/C. 2000.F. Phosphorylation of the human microRNA-generating complex mediates MAPK/Erk signaling. phosphatidylserine. et al. Exchange factors of the RasGRP family mediate Ras activation in the Golgi. Yan J. Uberall F. Yan H. Mol Cell Biol 28: 4377–85. et al. Mallabiabarrena A. Scaffold proteins of MAP-kinase modules. Harding A. Enrich C. Yizhar O. Matallanas D. Ding Q. Paterson H. Fincham VJ. Haklai R. Calvo F. Proc Natl Acad Sci USA 99: 7496–501. Canon E. et al. Sanz-Moreno V. Huber LA. Krueger J. 2004. 32. Teis D. et al. Ras isoform abundance and signalling in human cancer cell lines. Molecular mechanisms involved in Ras inactivation: the annexin A6-p120GAP complex. Mol Biol Cell 11: 2863–72. Paz A. Biophys J 92: 808–15. Omerovic J. Sobczyk A. PEA-15 mediates cytoplasmic sequestration of ERK MAP kinase. Rotblat B. Nuclear localization of the ERK MAP kinase mediated by Drosophila alphaPS2betaPS integrin and importin-7. 48. James M. Torii S. et al. 2001. 41. Associations of B. 37. Gonzalez JM. Teis D. Nature 427: 256–60. 1995. Theroux SJ. Elad-Sfadia G. Phospholipase Cg activates Ras on the Golgi apparatus by means of RasGRP1. et al. Matallanas D. Chen C. 2004. Bustelo XR. Bivona T. Jaumot M. Growth Factors 24: 21–44. Kurzbauer R. Arozarena I. 2007. The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. et al. 2008. Signaling crossroads: the function of Raf kinase inhibitory protein in cancer. Haklai R. Dev Cell 3: 803–14. 78. Nature 444: 724–9. et al. Sanz-Moreno V. Curr Opin Cell Biol 9: 180–6. 1997. Ballan E. Berciano MT. Acharya JK. Rotblat B. et al. 2006.
Walker J. Joe A. hSef inhibits PC-12 cell differentiation by interfering with Ras-mitogen-activated protein kinase MAPK signaling. Casar B. Cell 93: 605–15. Weinstein IB. 2002. Shaw AS. Hasan SS. Burack WR. Evans C. 84. Mebratu YA. Banu N. 93. J Cell Biol 148: 1267–81. The BH3-only protein Bik/ Blk/Nbk inhibits nuclear translocation of activated ERK1/2 to mediate IFNgamma-induced cell death. Catling AD. Chen CH. Ishibe S. J Cell Biol 183: 429–39. 1998. Live Cell Imaging of ERK and MEK: simple binding equilibrium explains the regulated nucleocytoplasmic distribution of ERK. Wang WJ. Science 281: 1668–71. Shenoy SK. Tohgo A. Ras subcellular localization deﬁnes extracellular signal-regulated kinase 1 and 2 substrate speciﬁcity through distinct utilization of scaffold proteins. 89. A constitutively active and nuclear form of the MAP kinase ERK2 is sufﬁcient for neurite outgrowth and cell transformation. et al. MP1: a MEK binding partner that enhances enzymatic activation of the MAP kinase cascade. Curr Biol 8: 1141–50.ß 2010 WILEY Periodicals. 2006. Zhao Q. 82. J Biol Chem 278: 19292–300. Burack WR. Bidirectional signals transduced by DAPK-ERK interaction promote the apoptotic effect of DAPK. Sanz-Moreno V. Le Mercier M. Mol Cell 12: 1275–85. 94. et al. The stability of the G protein-coupled receptor-beta-arrestin interaction determines the mechanism and functional consequence of ERK activation. 2003. Review essays Bioessays 32: 412–421. Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation. 92. Canagarajah B. Dickey BF. Differential expression of DHHC9 in microsatellite stable and instable human colorectal cancer subgroups. Wilsbacher J. Glycobiology 16: 137R–57R 96. Rong Z. 87. 2008. et al. EMBO J 24: 294–304. Glading A. et al. Zhu X. 91. Kinase suppressor of Ras (KSR) is a scaffold which facilitates mitogen-activated protein kinase activation in vivo. 81.. et al. 1998. Baker TL. et al. 2009. Receptor-speciﬁc ubiquitination of beta-arrestin directs assembly and targeting of seven-transmembrane receptor signalosomes. 2003. 2003. Cancer Res 67: 1536–44. Essential role of ERK dimers in the activation of cytoplasmic but not nuclear substrates by ERK-scaffold complexes. Galectins – potential targets for cancer therapy. Crespo P. 2007. et al. Zalevsky J. 99. Robinson MJ. 86. Cancer Lett 253: 25–33. PEA-15 inhibits tumor cell invasion by binding to extracellular signal-regulated kinase 1/2. J Biol Chem 280: 3832–7. Stock JL. 2005. Gesty-Palmer D. et al. Khokhlatchev AV. Beta-arrestindependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. Krueger J. Pinto A. et al. et al. 2008. Br J Cancer 96: 1896–903. Choy EW. Kuo JC. 1998. Stippec SA.. Kruhoffer M. 90. Casar B. Ashraf GM. J Biol Chem 280: 15315–24. 97. Oncogene addiction. Mol Cell Biol 29: 1338–53. 2008. Phosphorylation-dependent paxillinERK association mediates hepatocyte growth factor-stimulated epithelial morphogenesis. et al. Camby I. et al. Lefkowitz RJ. Arozarena I. Goldsmith E. 421 . Lefranc F. Nguyen A. Inc. Schaeffer HJ. Eblen ST. J Biol Chem 278: 6258–67. Cancer Res 68: 3077– 80. 2005. 2000. 95. et al. 83. 98. Prospects & Overviews F. 2005.. DeFea KA. J Biol Chem 278: 50273–82. 100. 2007. Distinct rates of palmitate turnover on membrane-bound cellular and oncogenic H-ras. 2003. Mol Cell Biol 22: 3035–45. Joly D. et al. Koziol JA. et al. Galectin-1: a small protein with major functions. 2007. Zheng H. Xiong S. Birkenkamp-Demtroder K. discussion 3080. Thoma MS. Mol Cell 31: 708–21. 88. Calvo et al. 85. Mansilla F.