Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mol Oncol. Author manuscript; available in PMC 2010 December 17.
Published in final edited form as:
PMCID: PMC3003591

Ras/MAPK Signaling from Endomembranes

Nicole Fehrenbacher,# Dafna Bar-Sagi,* and Mark Philips#¥


Signal transduction along the Ras/MAPK pathway has been generally thought to take place at the plasma membrane. It is now evident that the plasma membrane is not the only platform capable of Ras/MAPK signal induction. Fusion of Ras with green fluorescent protein and the development of genetically encoded fluorescent probes for Ras activation have revealed signaling events on a variety of intracellular membranes including endosomes, the Golgi apparatus and the endoplasmic reticulum. Thus, the Ras/MAPK pathway is spatially compartmentalized within cells and this may afford greater complexity of signal output.


The field of signal transduction concerns itself with the molecular mechanisms whereby biochemical signals are transduced across the plasma membrane and propagated within the cell in order to orchestrate a desired cellular response. More than any other field, signal transduction holds the promise of informing the process if drug discovery. Among the myriad of signaling molecules that have received great scrutiny in recent decades, perhaps none has been more intensely studied than Ras. For more than three decades great effort has been made to understand the intrinsic signaling properties of Ras proteins and the signaling networks regulated by them. The hope that the study of Ras signaling will lead to novel anti-cancer therapies has in no small part fueled the intense interest. For many years the study of Ras involved the “what and when” of signaling as investigators catalogued the upstream activators, negative regulators and downstream effectors of the GTPase and studied the kinetics of the Ras/MAPK pathway. Following from the discovery that Ras is expressed on several subcellular compartments and seeking to help explain the diversity of signal outputs emanating from a biochemically simple binary switch, Ras biologists have more recently focused on the “where” of signaling.

The plasma membrane (PM) is often considered the primary signaling platform because signaling complexes are assembled here when transmembrane receptors are engaged by extracellular ligands. Several sets of discoveries contributed to the initial assignment of Ras exclusively to the PM. First was the discovery that Ras proteins are peripheral membrane proteins localized on the inner leaflet of the plasma membrane [1]. Second was the discovery that Ras is associated with membranes by virtue of post-translational modification with lipids [2]. Finally, in genetic studies in flies, Ras was placed immediately downstream of growth factor receptors [3]. The revolution in cell biology ushered in by the age of green fluorescent protein (GFP) provoked a reassessment of the spatiotemporal aspects of Ras signaling. Using genetically encoded fluorescent probes, Ras signaling has been observed on intracellular membranes. In addition to the PM, Ras and/or MAPK signaling has now been observed on endosomes, the endoplasmic reticulum (ER), the Golgi apparatus, and mitochondria. Ras signaling from each of these platforms plays a role in the control a wide variety of cellular processes, including growth, survival and differentiation. Subcellular compartmentalization of signaling, such as that regulated by Ras, provides one explanation for the apparent complexity of signaling outputs elaborated by individual signaling molecules and, in the case of Ras, forms a framework for understanding the evolution of four isoforms that differ primarily in the way they are targeted to cellular membranes. In this review, we give an overview of current understanding of compartmentalized signaling focusing on the Ras/MAPK pathway.

Ras Biology - The GTPase, The Oncogene

Ras proteins are prototypical members of the superfamily of small GTPases. They transmit signals from cell surface receptors to a variety of effectors and thereby regulate pathways governing cell proliferation, differentiation, and programmed cell death [4]. Ras proteins act as molecular switches. Signal-induced conversion of the inactive to active state is mediated by guanine nucleotide-exchange factors (GEFs) that stimulate the exchange of GDP for GTP. This is accomplished by catalyzing the release of GDP from the guanine nucleotide binding pocket. Once nucleotide free, Ras next binds GTP because it is tenfold more abundant in cytosol than is GDP. A marked conformational change caused by GTP binding leads to activation of Ras [5]. The effector domain engages downstream signaling molecules only when the protein is in the GTP-bound state. The activation state of Ras is self-limited by the intrinsic GTPase activity of the protein. However, Ras, like most signaling GTPases, is a poor enzyme. The catalytic activity of Ras is greatly enhanced by GTPase activating proteins (GAPs). GEFs and GAPs thus cooperate to generate a critical level of regulation, allowing the signal to turn on and off and to persist for a relatively short but variable period of time. The availability of constitutively active as well as dominant-negative forms of Ras have made it possible to characterize its biological function [6]. The dominant-active forms are constitutively GTP-bound and mimic the oncogenic forms of the protein.

Ras genes encoded by rat sarcoma viruses, v-H-ras and v-K-ras, were among the first oncogenes to be recognized [7, 8]. These viral genes are mutant forms of cellular protooncogenes [911]. Mutations that render the GTPase insensitive to the action of GAP and therefore lock Ras in the GTP-bound state account for its oncogenic activity [12, 13]. Activated Ras alleles are the oncogene most frequently associated with human carcinomas [12], accounting for the great impact of Ras on human health. The mammalian genome encodes three ras genes that give rise to four gene products, N-Ras, H-Ras, K-Ras4A , and K-Ras 4B. All isoforms are ubiquitously expressed, although isoform ratios vary from tissue to tissue. K-ras4A and K-Ras4B are splice variants of the K-Ras gene that use alternative fourth exons. Mutations in ras genes are found in thirty percent of all human cancers. Tumors differ both in the isoforms associated with the disease and in the incidence of mutations of that isoform. For example, whereas 90% of pancreatic adenocarcinomas are associated with an oncogenic Ras mutation that is invariably in the K-Ras gene, only 10% of bladder carcinomas harbor Ras mutations, and these occur in the H-Ras gene [14].

Ras Processing and trafficking

Although distinct in trafficking and steady-state localization, one universal feature common to all Ras isoforms is that their localization on the cytosolic leaflet of cellular membranes is required for biological function. Evidence for Ras isoform-specific signaling has been provided from tumor profiling, knockout mice, and overexpression studies [15]. The subcellular compartmentalization of different Ras isoforms is partially overlapping, yet distinct, and accounts for their biological differences. Ras proteins begin their lives in the cytosol as globular hydrophilic proteins that display a C-terminal CAAX sequence (Figure 1). This sequence is the signal for a series of post-translational modifications that include farnesylation, AAX proteolysis and carboxyl methylation (reviewed in [16]). The enzymes that catalyze AAX removal, Rce1, and carboxyl methylation, Icmt, are restricted to the ER, which serves as a way station for nascent Ras [17, 18]. H-Ras, N-Ras and K-Ras4A are further modified with one or two palmitates [19]. The enzyme that palmitoylates Ras resides on the Golgi apparatus [20]. In contrast to the other isoforms, K-Ras4B is not palmitoylated and requires no further modification for full membrane affinity. Instead, K-Ras4B localization depends on a polybasic region immediately upstream of its C-terminal farnesyl cysteine [2]. Thus, K-Ras4B, hereafter referred to simply as K-Ras, is unique among the Ras isoforms and its subcellular trafficking is distinct. K-Ras is also the isoform most often associated with human cancer. Cancer biologists hope to exploit the unique features of K-Ras trafficking to develop new anti-Ras drugs.

Figure 1
Post-translational Modification and Trafficking of Ras

As peripheral membrane proteins, Ras proteins have two ways by which they can move from one membrane compartment to another. First, they can travel like intrinsic membrane proteins that are transferred from compartment to compartment via vesicular transport. Second, they can detach from the donor membrane and move through the aqueous phase of the cytosol, with or without a chaperone to shield their farnesyl chain, to the acceptor membrane. Significant evidence exists for each mode of transport. GFP-tagged Ras proteins can be readily observed on highly motile vesicles, some of which travel along microtubules in a linear, saltatory fashion [21]. N-Ras and H-Ras have been found to undergo a palmitoylaton/depalmitoylation cycle whereby depalmitoylation favors release of the GTPases from the inner leaflet of the PM from whence they travel retrograde, via diffusion through the cytosol, to the Golgi apparatus. Upon arrival at the Golgi, Ras proteins are replamitloylated and thereby once again affinity trapped in the membrane and sent back to the PM by vesicular transport [22, 23]. Because K-Ras associates with the PM via an intrinsic polybasic sequence rather than a labile palmitate modification, on first principles one might assume that its membrane association is constitutive. Elegant studies by Silvius showed this is not the case. Rather, PM K-Ras is in a dynamic equilibrium with a pool in the cytosol [24]. More recently, the association of K-Ras with the inner leaflet of the PM has been shown to be regulated by calmodulin binding to the polybasic region [25] and by phosphorylation by PKC of serine 181 within the polybasic region (see below) [26].

Compartmentalization of Ras isoforms does not end with delivery of the mature proteins to the PM. A large amount of evidence has been generated over the past decade that demonstrates that H-Ras and K-Ras reside in distinct microdomains within the PM, the former partitioning into lipid ordered domains high in cholesterol and sometimes referred to as lipid rafts and the latter restricted to disordered domains [27]. Remarkably, the partition of Ras proteins into microdomains, referred to as nanoclusters, is regulated by the GTP/GDP state of the protein suggesting that either the G domain participates in membrane association or this region controls the conformation of the hypervariable C-terminus in such a way as to affect microdomain partition [28]. The influence of PM microdomain localization on Ras signaling may be as profound as that of organelle localization [29]. Indeed, Hancock and colleagues have put forth elegant models demonstrating how nanoclusters of Ras can transform analog signal input into digital output [30]. However, this area has been extensively reviewed in recent years [31] and we will focus here on subcellular localization at the level of the organelle.

GFP-tagged Ras proteins have been visualized on PM, Golgi apparatus, ER, mitochondria and a variety of endosomes. Ras isoforms display different degrees of association with endomembranes; N≥H>K-Ras [21]. These observations raise the obvious question of whether endomembrane associated Ras is capable of signaling and, perhaps more important, whether the signal output varies depending on the subcellular platform? Fluorescent probes of Ras activity have been used to address this question and the answer appears to be, yes; Ras can become activated on endomembrane and send a signal down various pathways and the outcome and duration of signaling depends, to some extent, on location [32].

Ras Signalling

The best-characterized signaling pathway regulated by Ras is the MAPK pathway that proceeds through Erk1 and Erk2. After growth factors bind to their cognate protein tyrosine kinase receptors (PTKRs), these receptors dimerize, which in turn allows cross-phosphorylation of tyrosine residues in their cytosolic domains catalyzed by the intrinsic kinase domain [3]. Among the signaling molecules that bind to phosphotyrosines on the cytosolic domains of these receptors is the adaptor Grb2 that binds SOS, which in turn acts as GEF for Ras proteins. Thus, phosphorylation of PTKRs leads to the recruitment of SOS to the PM where it can encounter Ras. GTP-bound Ras then recruits the Raf-1 kinase. The molecular details of the kinase activation of Raf-1 remains poorly understood but require membrane association of Ras [33]. Raf-1 subsequently phosphorylates and activates MEK (MAPK/Erk kinase), a dual specificity tyrosine/threonine kinase, that, in turn, phosphorylates and activates Erk1 and Erk2. The latter are serine/threonine kinases with numerous substrates, both nuclear and cytoplasmic. Phospho-Erk forms dimers that translocate to the nucleus, where they phosphorylate the Ets family of transcription factors, including Elk-1. In this way a signal originating from an extracellular stimulus is transmitted from the cell surface to the nucleus where gene transcription is modulated.

The Raf-1/Erk pathway is not the only one regulated by Ras. Ras effectors can be defined as those proteins that bind to Ras via its effector domain only when Ras is GTP-bound. More stringently defined, the function of the effector must be modulated by the binding to GTP-Ras. Today, more than ten types of proteins are characterized as putative Ras effectors [34]. Apart from Raf-1, the best characterized effectors are phosphatidylinositol 3-kinase (PI3K) and members of a family of exchange factors for the small GTPase Ral, e.g. RalGDS. Although the Raf-1/MAPK pathway has been defined as sufficient for Ras-mediated transformation of mouse fibroblasts, recent data have suggested that in human cells the RalGDS pathway is the most important for transformation [35]. In addition, PI3K activates a pro-survival kinase is Akt/PKB, which in some circumstances may be essential for oncogenesis [36].

Ras signaling on the ER and Golgi

GFP fusions with Ras proteins established that Ras transits the ER en route to the PM and that at steady state palmitoylated isoforms of Ras are expressed on the Golgi apparatus as well as the PM [21, 37]. The first indication that intracellular Ras was capable of activation downstream of growth factor signaling came from studies using GFP fused to the Ras binding domain (RBD) of Raf-1 [32]. At about the same time, Matsuda designed innovative FRET activation probes for Ras family proteins that he designated RAICHU. Using RAICHU-Ras Matsuda reported that activation was limited to the PM [38]. However, RAICHU-Ras was targeted to membranes using the C-terminus of K-Ras4B such that it bypassed the Golgi and therefore did not accurately report spatiotemporal signaling of palmitoylated Ras isoforms. Using GFP-RBD probes that are spatially unbiased it is now well established that GTP-bound Ras accumulates on the Golgi as well as the PM in response to growth factor signaling in fibroblasts and TCR signaling in lymphocytes [23, 32, 3941].

GFP-RBD probes report Ras activation by simple translocation. Their biggest drawback is that they are not sensitive enough to report activation of endogenous Ras and co-expression of wild-type Ras isoforms is required in live cell assays. However, using FRET based analysis, Chiu et al. confirmed that the results observed with GFP-RBD expressed with untagged, wild-type H-Ras are identical to those seen with endogenous Ras [32, 39]. Augsten and colleagues have recently introduced a trimeric GFP-RBD probe that contains three tandem, doubly mutated RBD domains because the equivalent probe with native domains proved to be highly toxic. These investigators claim that their probe is sensitive enough to report activation of endogenous Ras, which they detect only on the PM of Jurkat T cells stimulated with PMA and ionomycin [42]. Physiologic signaling through the TCR was not reported. Thus, although there is some controversy in the field, there remains a preponderance of evidence supporting the idea that GTP-bound palmitoylated Ras accumulates on the Golgi downstream of receptor signaling.

Although Ras activation on the Golgi has been reported in a number of cell types it has been most extensively studied in T lymphocytes downstream of TCR signaling. This is due to several features specific to T cells. First, although T cells express both N-Ras and K-Ras it appears to be N-Ras that is most important for T cell activation [43] and when hematopoetic malignancies are associated with a mutated ras gene it is most often nras. Second, whereas GFP-RBD recruitment to the Golgi in fibroblasts can take more than 20 min of growth factor signaling [32], the same phenomenon in T cells stimulated through the TCR can be detected in under 2 min [39]. Finally, RasGRP1, the exchange factor associated with Ras activation on the Golgi is highly expressed in T cells. Spatiotemporal Ras signaling has proven to be very interesting in T lymphocytes. When the TCR alone was engaged there was a rapid and robust accumulation of GTP-Ras on the Golgi without any detectable signal at the PM [39, 41]. However, when more physiologic signaling was initiated by simultaneous ligation of both the TCR and the LFA-1 co-receptor, robust accumulation of GTP-Ras on the PM was observed in addition to a pool activated on the Golgi. Both pools of GTP-bound Ras were controlled by RasGRP1 that gained access to the PM following LFA-1 engagement because the integrin co-receptor activated phospholipase D2 to generate phosphatidic acid which was then converted to DAG [41]. Thus, the combination of receptors engaged on T lymphocytes determines the spatiotemporal features of Ras signaling.

If GTP-bound Ras accumulates on the Golgi there are two mechanisms that may drive this process. First, Ras could be activated at the PM and traffic to the Golgi apparatus in a retrograde pathway following depalmitoylation at the PM. This model is favored by Bastieans and colleagues who showed, using photoactivatable GFP, that H-Ras and N-Ras traffic retrograde from PM to Golgi via a non-vesicular, diffusion limited pathway [23], a conclusion supported by the FRAP studies of Kenworthy [22]. This model requires a relatively long half-life of GTP binding suggesting a lack of access to GAPs during depalmitoylation at the PM, cytosolic transport and accumulation on the Golgi. An alternative, model holds that H-Ras and N-Ras can be activated in situ on the Golgi. This model is made quite plausible in lymphocytes where RasGRP1 is regulated by diacylglycerol and calcium and has affinity for the Golgi following TCR engagement [39, 41]. Of course, these two models are not mutually exclusive and GTP-bound Ras on the Golgi may derive from both pathways. In support of the idea that Ras exchange factors preferentially activate spatially restricted pools of Ras, Arozarena et al. reported that RasGRF activated H-Ras on the ER [44].

Perhaps more relevant that GTP-bound Ras on the Golgi or ER is the question of whether activated Ras so situated can signal to downstream effectors and, if so, are these signals qualitatively different than those emanating from the PM? To address this question Chiu et al. stringently targeted constitutively activated Ras proteins to various subcellular membranes using a variety of transmembrane segments fused to the N-terminus of Ras. H-Ras61L potently transformed rodent fibroblasts when restricted to either the ER or Golgi [32, 39]. Importantly, when signaling output to three biochemical pathways was compared following expression of endomembrane-tethered Ras, Erk and Akt were as efficiently activated from the Golgi as from the PM but Jnk was not. In contrast, activated Ras restricted to the ER was very efficient in activating Jnk providing a compelling demonstration of compartmentalized signaling [32]. Crespo and colleagues took a similar approach and found preferential signaling down the RalGDS pathway from Golgi tethered Ras and robust signaling to Erk, Akt and Jnk from ER restricted Ras [45]. Although these investigators found little Erk phosphorylation induced by Golgi-tethered Ras, Elk-1 phosphorylation and luciferase activity was nevertheless stimulated equally well from this compartment [46]. Thus, although some results differ, the idea of compartmentalized signaling to specific effectors is supported by both studies.

Artificially targeting Ras to various organelles has firmly established that signaling is possible from these locations but does not establish that such compartmentalized signaling occurs in vivo. One advance toward establishing such a model in vivo comes from genetic studies of fission yeast. In S. pombe a single Ras protein, Ras1p, controls at least two distinct pathways, that regulating cell morphology and that regulating mating. Onken et al. showed that these distinct pathways are regulated by Ras1p in different locations; signals emanating from the ER controlled cell morphology and those originating at the PM drove mating [47]. The most compelling data to date for a biological role for compartmentalized Ras signaling comes from the study of T lymphocytes. Palmer and colleagues have explained a long baffling paradox of lymphocyte signaling by spatiotemporal signaling of Ras. Positive versus negative selection of thymocytes represent diametrically opposed processes leading, respectively, to proliferation or programmed cell death. Yet the Ras/MAPK pathway is required for both biological outcomes. Daniels et al. showed that whereas strong antigens that stimulate negative selection activate the Ras/MAPK pathway at the PM, weaker antigens that induce positive selection initiate signaling from the Golgi [48]. Thus the biological outcome of Ras signaling can depend on the subcellular platform from which signaling is initiated.

Ras on Mitochondria

All three Ras isoforms have been found associated with subcellular fractions enriched in mitochondria [49, 50]. In contrast, numerous studies utilizing live cell imaging with GFP-tagged Ras proteins failed to detect Ras on mitochondria despite unambiguous expression on ER, Golgi and a variety of vesicles. One explanation for this discrepancy is that localization of Ras by subcellular fractionation is complicated by ex vivo release of Ras from membranes decorated with the protein in intact cells and subsequent non-specific adsorption of the Ras onto membranes and organelles in the cellular homogenate, a phenomenon that is particularly problematic with K-Ras that undergoes non-specific electrostatic interactions via its C-terminal polybasic region. More compelling evidence for mitochondrial association of Ras was provided by Bivona et al. who reported GFP-K-Ras translocation to the mitochondrial outer membrane in living cells treated with the PKC agonist bryostatin-1 [26]. A farnesyl-electrostatic switch involving phosphorylation of serine 181 in the polybasic region serves to partially neutralize the electrostatic charge and promote release from the PM. Surprisingly, phospho-K-Ras was associated with programmed cell death and Bcl-Xl was required for induction of apoptosis. Like phospho-K-Ras, Bcl-Xl resides on the mitochondrial outer membrane where it interacts with K-Ras. Thus, K-Ras signaling from mitochondria may not only engage distinct effectors but may have a biological outcome (cell death) diametrically opposed to Ras signaling from other membranes (proliferation and survival). It may be possible to exploit this feature of K-Ras signaling to develop anti-cancer drugs.

Ras Signalling on endosomes

Because endosomes derive from the PM and are well known to internalize PTKRs it is not surprising that Ras signaling has been reported on endosomes. Indeed, endosomes serve as the most diverse and dynamic endomembrane compartment upon which Ras has been found to reside and signal. Originally conceived of as a process that limits signaling by removing receptors from the surface, several groups have subsequently shown that, in many contexts, efficient growth factor signaling requires endocytosis [51]. The first evidence for endosomal signaling came from subcellular fractionation studies in which Shc, Grb2, mSOS, and phospho-Raf-1 were differentially observed on endosomes following EGF or insulin stimulation [52]. Subsequently inhibition of clathrin-mediated endocytosis with agents such as dominant negative dynamin (K44A) proved to inhibit rather than enhance Ras/MAPK signaling [53, 54]. Endosomal signaling may affect the kinetics of signaling: growth factor stimulated MAPK activity has been reported to be transient from the PM and sustained from endosomes [55, 56].

Several methods have been used to demonstrate that PTKRs remain active upon internalization on endosomes. EGF remains bound to its receptor in early endosomes and the EGF receptor (EGFR) itself is phosphorylated after internalization [57]. In addition to phosphorylated, active PTKRs, other upstream components of Ras signaling have been localized on endosomes, including Shc, Grb2, SOS, and PLCγ1 [32, 52, 55, 5762]. Ras itself was observed on endosomes using subcellular fractionation [63]. Using GFP fusion proteins and live cell imaging, Ras has been localized to vesicles, including endosomes [21, 58]. Sorkin and colleagues have used YFP-RBD to reveal GTP-bound Ras on vesicles decorated with internalized EGF [58]. Roy et al. showed that H-Ras but not K-Ras signaling requires endocytosis [64] and this result was confirmed by Omerovic et al. who found that N-Ras signaling also required endocytosis [65].

Piertro De Camilli and colleagues recently provided a major advance in endosomal signaling when they showed that signaling endosomes characterized by association of the adaptor protein APPL1 mature into EEA1 positive early endosomes by accumulation of PI3P and can be reverted by hydrolysis of the phosphate at the 3 position [66], (Fig. 2). Importantly, reversion of early endosomes enhanced growth factor signaling demonstrating that the APPL1 positive compartment is particularly adapted for signaling. Thus, the complexity of endosomal signaling is greater than previously appreciated. If one takes into account that signaling has been observed from both clathrin-dependent and independent endosomes, the complexity of the system increases even more.

Figure 2
Ras Signaling from Endomembranes

Internalized receptors are either further trafficked to lysosomes for degradation or recycled back to the PM [67]. A recent study by Lu et al. adds late endosomes and lysosomes to the pantheon of membrane platforms from which K-Ras signals (Fig. 2). In this study EGFR signaling was associated with the progressive accumulation of GFP-K-Ras but not N-Ras or H-Ras on early EEA1/Rab5 bearing endosomes, Rab7-marked endosomes, LAMP1/2- marked lysosomes and multivesicular bodies (MVBs)[68]. Furthermore, late endosomes, lysosomes and MVBs were shown to serve not only as platforms for Ras signaling but also as sites for K-Ras degredation. K-Ras was stabilized by inhibitors of lysosomal degradation but not by proteasome inhibitors [68]. This latter observation is somewhat surprising given the disposition of K-Ras and other Ras proteins to the cytosol. The occurrence of large multivesicular structures decorated with GFP-Kras, as seen by Lu et al., could be the result of the long-term incubation with protease inhibitors employed in these studies. Protease inhibitors are known to induce autophagosome formation, perhaps through a lysosomal stress pathway [69]. Furthermore, because protease inhibitors upregulate the lysosomal membrane proteins, LAMP-1/2 [70], the increased co-localization of K-Ras with LAMP-1/2 observed by Lu et al. may also be a consequence of protease inhibition.

It remains to be clarified whether K-Ras association with endosomes is a result of clathrin-mediated endocytosis of membranes carrying K-Ras, or if K-Ras arrives on these compartments by translocation through the cytosol, or both. Regardless of the mechanism of trafficking to late endosomes, this results of Lu et al. contradicts earlier studies from several groups that found that whereas N-Ras and H-Ras trafficked stably associate with endosomes K-Ras does not [64, 65, 71]. This differential trafficking pattern has been attributed to the preferential modification of H- and N-Ras by non-degradative mono and Lys 63-linked di ubiquitination. The selective targeting of H- and N-Ras for ubiquitination is determined by their C-terminal membrane targeting regions likely reflecting the confinement of the enzymatic machinery that controls Ras ubiquitination to specific membrane compartments either at the level of organelles or membrane nanodomains. Similarly to other membrane cargo proteins, the ubiquitination of H- and N-Ras promotes their association with endosomes (Fig. 2). As a consequence, the pool of PM-associated Ras molecules that is available for the recruitment and activation of Raf-1 is reduced and ERK activation is attenuated [71]. Consistent with these findings, it has been recently shown that that in Drosophila, maintaining a threshold of Ras ubiquitination is critical to prevent inappropriate Ras-ERK activation in vivo [72]. Thus, rather than functioning as signaling endosomes, the pool of endosomes bearing ubiquitinated Ras may not be capable of signaling and may thereby afford a mechanism for downregulation of Ras/MAPK signaling.

Other recent findings shed some doubt on the idea of endosomes as fully competent MAPK signaling platforms. MEK2-GFP was observed both on the PM and endosomes but the activated form of the kinase was detected only on the PM [73]. Moreover, the population of endosomes decorated with MEK2-GFP was distinct from those that carried activated EGFR. Interestingly, in this study silencing of clathrin heavy chain augmented EGF stimulation of Erk, a result inconsistent with studies using dominant negative dynamin as a way of blocking endocytosis [51]. Thus it is clear that the complexity and physiologic relevance of signaling from endosomes remains to be fully elucidated.

MAPK Scaffolds

Compartmentalized Ras signaling is facilitated in part by the differential subcellular trafficking of Ras isoforms. However, Ras localization is not the only mechanism for compartmentalization. An increasingly recognized component of MAPK and other signaling pathways are scaffold proteins that play no direct role in catalysis but serve as platforms upon which signaling complexes can assemble. Because many scaffolds are spatially restricted within cells this class of molecule can contribute to and, in some cases define, compartmentalized signaling. Scaffolding molecules in the MAPK pathways serve to organize the various MAPK modules, such as Erk, Jnk and p38 [74]. A well-studied Erk scaffold is the kinase suppressor of Ras (KSR), a protein poorly named since it is not a kinase and was first identified in screens in flies [75] and worms [76, 77] as a positive regulator of the Ras/MAPK pathway. KSR is a multidomain protein that binds Raf-1, MEK and Erk, as well as other proteins. Like Raf-1, KSR is sequestered in the cytosol by 14-3-3 proteins in resting cells. Upon mitogenic stimulation KSR translocates to the PM when it becomes dephosphorylated at serine 392 and looses its affinity for 14-3-3 [78, 79]. Thus, KSR serves as inducible scaffold for Ras/MAPK signaling with specificity for the PM. Recently the specificity of KSR has been further resolved to the nanoscale by a study that reports that the scaffolding activity of KSR1 is specific to lipid microdomains within the PM [46]. Interestingly, in this study IQGAP1 also served as a lipid-raft specific MAPK scaffold that promoted phosphorylation of EGFR [46].

MEK partner 1 (MP1) was first identified in a yeast two-hybrid screen as a binding partner of MEK1 [80]. It preferentially binds to MEK1 and Erk1, not MEK2 and Erk2. Binding facilitated the phosphorylation of Erk1 by MEK1, thus fulfilling the criteria for a scaffold [80]. Interestingly, MP1 was also found to interact with p14, a highly conserved protein that resides on the cytoplasmic face of early endosomes [81]. Overexpression studies revealed that MP1 increases Erk signaling, but only when overexpressed with p14. Using the C-terminus of K-Ras, the MP1/p14 complex was ectopically targeted to the PM. This construct failed to augment Erk activation, indicating that the endosomal location is essential for the scaffolding function of MP1/p14 [81]. Thus, MP1 is an endosome-specific scaffold for MEK and Erk. Interestingly, while MP1/p14 was not required for early activation at the PM, it was required for the activation seen on endosomes 10–30 min after EGF stimulation [81].

MP1 is not the only MAPK scaffold on endosomes; β-arrestin also provides this function in GPCR signaling. This multifunctional protein regulates internalization of GPCRs into clathrin-coated vesicles [82, 83] and serves as a scaffold for both the Erk [84] and Jnk [85] MAPK modules. Several studies place β-arrestin functionally in the transmission of signals from Raf-1 to MEK and Erk on endosomes. Raf-1 overexpression increased MEK and Erk binding to β-arrestin [84] and a dominant-negative form of β-arrestin blocked Erk activation downstream of a GPCR [8688]. Thus, endosomes are a subcellular site that supports both PTKR and GPCR signaling to Erk, and each system uses distinct scaffolds on this organelle.

Sef is yet another MEK/Erk scaffold that affords spatial specificity. Sef resides on the Golgi apparatus [89]. Sef was originally identified as a negative regulator of fibroblast growth factor [90, 91]. Sef binds MEK only when the kinase is phosphorylated and activated. In the canonical MAPK cascade Erk dissociates from MEK once it is phosphorylated forms dimers that subsequently enters the nucleus. However, activated Erk remains associated with MEK on Sef, preventing Erk’s translocation into the nucleus and preventing is interaction with nuclear substrates such as Elk-1. Nevertheless, active Erk associated with Sef on the Golgi is capable of phosphorylating cytosolic substrates such as RSK2 [89]. When Elk-1 was tagged with a nuclear export signal, it became artificially localized to the cytoplasm and became a substrate for Sef-associated phospho-Erk [89]. This makes Sef a compartment-specific scaffold protein that directs Erk activity to one set of substrates over another [92]. Recently, Sef was reported to be required for Ras signaling from the ER [46]. Like Sef, β-arrestin 1 can also shift the substrate specificity of Erk during GPCR signaling such that cytosolic substrates are favored and cell proliferation is not triggered [88]. Similarly, β-arrestin 2 acts to sequester Jnk3 in the cytosol [85]. Thus the spatiotemporal controls of Ras/MAPK signaling imparted by the various scaffolds translate into substrate specificity and therefore pathway selection.

Concluding Remarks

One of the central paradoxes of signal transduction is how a signaling molecule such as Ras, which appears from a biochemical point of view to be a simple binary switch, can independently regulate so many different pathways. One way to enhance the complexity of Ras signaling is to by compartmentalization within cells. The nature of Ras as a peripheral membrane protein conditionally associated with the cytoplasmic leaflet of cellular membranes allows for a diversity of localizations on various organelles and, taken to the nanoscale, in a variety of membrane microdomains. The peripatetic nature of Ras allows for compartmentalized signaling. Indeed, Ras/MAPK signaling has now been established on PM, endosomes, Golgi and ER and K-Ras signals from the surface of mitochondria. In the case of T lymphocytes it appears that the location of Ras/MAPK signaling dictates the biological outcome. As new tools are developed to allow measurement of compartmentalized Ras/MAPK signaling in live cells and in vivo we are bound to learn more about how location dictates function.


Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


1. Willingham MC, Pastan I, Shih TY, Scolnick EM. Localization of the src gene product of the Harvey strain of MSV to plasma membrane of transformed cells by electron microscopic immunocytochemistry. Cell. 1980;19:1005–1014. [PubMed]
2. Hancock JF, Paterson H, Marshall CJ. A polybasic domain or palmitoylation is required in addition to the CAAX motif to loacalize p21ras to the plasma membrane. Cell. 1990;63:133–139. [PubMed]
3. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2000;103:211–225. [PubMed]
4. Karnoub AE, Weinberg RA. Ras oncogenes: split personalities. Nat Rev Mol Cell Biol. 2008;9:517–531. [PMC free article] [PubMed]
5. Vetter IR, Wittinghofer A. The guanine nucleotide-binding switch in three dimensions. Science. 2001;294:1299–1304. [PubMed]
6. Feig LA. Tools of the trade: use of dominant-inhibitory mutants of Ras-family GTPases. Nat Cell Biol. 1999;1:E25–E27. [PubMed]
7. Harvey JJ. An Unidentified Virus Which Causes the Rapid Production of Tumours in Mice. Nature. 1964;204:1104–1105. [PubMed]
8. Kirsten WH, Mayer LA. Malignant lymphomas of extrathymic origin induced in rats by murine erythroblastosis virus. J Natl Cancer Inst. 1969;43:735–746. [PubMed]
9. DeFeo D, Gonda MA, Young HA, Chang EH, Lowy DR, Scolnick EM, Ellis RW. Analysis of two divergent rat genomic clones homologous to the transforming gene of Harvey murine sarcoma virus. Proceedings of the National Academy of Sciences of the United States of America. 1981;78:3328–3332. [PubMed]
10. Ellis RW, DeFeo D, Shih TY, Gonda MA, Young HA, Tsuchida N, Lowy DRS. The p21 src genes of Harvey and Kirsten sarcoma viruses originate from divergent members of a family of normal vertebrate genes. Nature. 1981;292:506–511. [PubMed]
11. Ruta M, Wolford R, Dhar R, Defeo-Jones D, Ellis RW, Scolnick EM. Nucleotide sequence of the two rat cellular rasH genes. Molecular & Cellular Biology. 1986;6:1706–1710. [PMC free article] [PubMed]
12. Barbacid M. ras Genes. Annual Review of Biochemistry. 1987;56:779–827. [PubMed]
13. Lowy DR, Willumsen BM. Function and regulation of ras. Annual Review of Biochemistry. 1993;62:851–891. [PubMed]
14. Bos JL. ras oncogenes in human cancer: a review. Cancer Res. 1989;49:4682–4689. [PubMed]
15. Hancock JF. Ras proteins: different signals from different locations. Nat Rev Mol Cell Biol. 2003;4:373–384. [PubMed]
16. Mor A, Philips MR. Compartmentalized Ras/MAPK Signaling. Annu Rev Immunol. 2006 [PubMed]
17. Boyartchuk VL, Ashby MN, Rine J. Modulation of Ras and a-Factor Function by Carboxyl-Terminal Proteolysis. Science. 1997;275:1796–1800. [PubMed]
18. Dai Q, Choy E, Chiu V, Romano J, Slivka S, Steitz S, Michaelis S, Philips MR. Mammalian prenylcysteine carboxyl methyltransferase is in the endoplasmic reticulum. Journal of Biological Chemistry. 1998;273:15030–15034. [PubMed]
19. Hancock JF, Magee AI, Childs JE, Marshall CJ. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989;57:1167–1177. [PubMed]
20. Swarthout JT, Lobo S, Farh L, Croke MR, Greentree WK, Deschenes RJ, Linder ME. DHHC9 and GCP16 constitute a human protein fatty acyltransferase with specificity for H- and N-Ras. J Biol Chem. 2005;280:31141–31148. [PubMed]
21. Choy E, Chiu VK, Silletti J, Feoktistov M, Morimoto T, Michaelson D, Ivanov IE, Philips MR. Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi. Cell. 1999;98:69–80. [PubMed]
22. Goodwin JS, Drake KR, Rogers C, Wright L, Lippincott-Schwartz J, Philips MR, Kenworthy AK. Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway. J Cell Biol. 2005;170:261–272. [PMC free article] [PubMed]
23. Rocks O, Peyker A, Kahms M, Verveer PJ, Koerner C, Lumbierres M, Kuhlmann J, Waldmann H, Wittinghofer A, Bastiaens PI. An Acylation Cycle Regulates Localization and Activity of Palmitoylated Ras Isoforms. Science. 2005;307:1746–1752. [PubMed]
24. Silvius JR, Bhagatji P, Leventis R, Terrone D. K-ras4B and Prenylated Proteins Lacking "Second Signals" Associate Dynamically with Cellular Membranes. Mol Biol Cell. 2006;17:192–202. [PMC free article] [PubMed]
25. Fivaz M, Meyer T. Reversible intracellular translocation of KRas but not HRas in hippocampal neurons regulated by Ca2+/calmodulin. J Cell Biol. 2005;170:429–441. [PMC free article] [PubMed]
26. Bivona TG, Quatela SE, Bodemann BO, Ahearn IO, Soskis MJ, Mor A, Miura J, Wiener HH, Wright L, Saba SG, et al. PKC regulates a farnesyl-electrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis. Mol Cell. 2006;21:481–493. [PubMed]
27. Hancock JF, Parton RG. Ras plasma membrane signaling platforms. Biochem J. 2005;389:1–11. [PubMed]
28. Prior IA, Harding A, Yan J, Sluimer J, Parton RG, Hancock JF. GTP-dependent segregation of H-ras from lipid rafts is required for biological activity. Nat Cell Biol. 2001;3:368–375. [PubMed]
29. Inder K, Harding A, Plowman SJ, Philips MR, Parton RG, Hancock JF. Activation of the MAPK module from different spatial locations generates distinct system outputs. Mol Biol Cell. 2008;19:4776–4784. [PMC free article] [PubMed]
30. Tian T, Harding A, Inder K, Plowman S, Parton RG, Hancock JF. Plasma membrane nanoswitches generate high-fidelity Ras signal transduction. Nat Cell Biol. 2007;9:905–914. [PubMed]
31. Henis YI, Hancock JF, Prior IA. Ras acylation, compartmentalization and signaling nanoclusters (Review) Mol Membr Biol. 2009;26:80–92. [PMC free article] [PubMed]
32. Chiu VK, Bivona T, Hach A, Sajous JB, Silletti J, Wiener H, Johnson RL, Cox AD, Philips MR. Ras signalling on the endoplasmic reticulum and the Golgi. Nat Cell Biol. 2002;4:343–350. [PubMed]
33. Morrison DK, Cutler RE. The complexity of Raf-1 regulation. Curr Opin Cell Biol. 1997;9:174–179. [PubMed]
34. Repasky GA, Chenette EJ, Der CJ. Renewing the conspiracy theory debate: does Raf function alone to mediate Ras oncogenesis? Trends Cell Biol. 2004;14:639–647. [PubMed]
35. Hamad NM, Elconin JH, Karnoub AE, Bai W, Rich JN, Abraham RT, Der CJ, Counter CM. Distinct requirements for Ras oncogenesis in human versus mouse cells. Genes Dev. 2002;16:2045–2057. [PubMed]
36. Gupta S, Ramjaun AR, Haiko P, Wang Y, Warne PH, Nicke B, Nye E, Stamp G, Alitalo K, Downward J. Binding of ras to phosphoinositide 3-kinase p110alpha is required for ras-driven tumorigenesis in mice. Cell. 2007;129:957–968. [PubMed]
37. Apolloni A, Prior IA, Lindsay M, Parton RG, Hancock JF. H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway. Mol Cell Biol. 2000;20:2475–2487. [PMC free article] [PubMed]
38. Mochizuki N, Yamashita S, Kurokawa K, Ohba Y, Nagai T, Miyawaki A, Matsuda M. Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature. 2001;411:1065–1068. [PubMed]
39. Bivona TG, Perez De Castro I, Ahearn IM, Grana TM, Chiu VK, Lockyer PJ, Cullen PJ, Pellicer A, Cox AD, Philips MR. Phospholipase Cgamma activates Ras on the Golgi apparatus by means of RasGRP1. Nature. 2003;424:694–698. [PubMed]
40. Caloca MJ, Zugaza JL, Matallanas D, Crespo P, Bustelo XR. Vav mediates Ras stimulation by direct activation of the GDP/GTP exchange factor Ras GRP1. Embo J. 2003;22:3326–3336. [PubMed]
41. Mor A, Campi G, Du G, Zheng Y, Foster DA, Dustin ML, Philips MR. The lymphocyte function-associated antigen-1 receptor costimulates plasma membrane Ras via phospholipase D2. Nat Cell Biol. 2007;9:713–719. [PubMed]
42. Augsten M, Pusch R, Biskup C, Rennert K, Wittig U, Beyer K, Blume A, Wetzker R, Friedrich K, Rubio I. Live-cell imaging of endogenous Ras-GTP illustrates predominant Ras activation at the plasma membrane. EMBO Rep. 2006;7:46–51. [PubMed]
43. Pérez de Castro I, Bivona T, Philips M, Pellicer A. Ras activation in Jurkat T cells following low-grade stimulation of the T-cell receptor is specific to N-Ras and occurs only on the Golgi. Mol Cell Biol. 2004;24:3485–3496. [PMC free article] [PubMed]
44. Arozarena I, Matallanas D, Berciano MT, Sanz-Moreno V, Calvo F, Munoz MT, Egea G, Lafarga M, Crespo P. Activation of H-Ras in the endoplasmic reticulum by the RasGRF family guanine nucleotide exchange factors. Mol Cell Biol. 2004;24:1516–1530. [PMC free article] [PubMed]
45. Matallanas D, Sanz-Moreno V, Arozarena I, Calvo F, Agudo-Ibanez L, Santos E, Berciano MT, Crespo P. Distinct Utilization of Effectors and Biological Outcomes Resulting from Site-Specific Ras Activation: Ras Functions in Lipid Rafts and Golgi Complex Are Dispensable for Proliferation and Transformation. Mol Cell Biol. 2006;26:100–116. [PMC free article] [PubMed]
46. Casar B, Arozarena I, Sanz-Moreno V, Pinto A, Agudo-Ibanez L, Marais R, Lewis RE, Berciano MT, Crespo P. Ras subcellular localization defines extracellular signal-regulated kinase 1 and 2 substrate specificity through distinct utilization of scaffold proteins. Mol Cell Biol. 2009;29:1338–1353. [PMC free article] [PubMed]
47. Onken B, Wiener H, Philips M, Chang EC. Compartmentalized signaling of Ras in fission yeast. Proc Natl Acad Sci U S A. 2006;103:9045–9050. [PubMed]
48. Daniels MA, Teixeiro E, Gill J, Hausmann B, Roubaty D, Holmberg K, Werlen G, Hollander GA, Gascoigne NR, Palmer E. Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling. Nature. 2006;444:724–729. [PubMed]
49. Rebollo A, Perez-Sala D, Martinez AC. Bcl-2 differentially targets K-, N-, and H-Ras to mitochondria in IL-2 supplemented or deprived cells: implications in prevention of apoptosis. Oncogene. 1999;18:4930–4939. [PubMed]
50. Wolfman JC, Planchon SM, Liao J, Wolfman A. Structural and functional consequences of c-N-Ras constitutively associated with intact mitochondria. Biochim Biophys Acta. 2006;1763:1108–1124. [PubMed]
51. Di Fiore PP, De Camilli P. Endocytosis and signaling. An inseparable partnership. Cell. 2001;106:1–4. [PubMed]
52. Di Guglielmo GM, Baass PC, Ou WJ, Posner BI, Bergeron JJ. Compartmentalization of SHC, GRB2 and mSOS, and hyperphosphorylation of Raf-1 by EGF but not insulin in liver parenchyma. Embo J. 1994;13:4269–4277. [PubMed]
53. Kranenburg O, Verlaan I, Moolenaar WH. Dynamin is required for the activation of mitogen-activated protein (MAP) kinase by MAP kinase kinase. J Biol Chem. 1999;274:35301–35304. [PubMed]
54. Vieira AV, Lamaze C, Schmid SL. Control of EGF receptor signaling by clathrin-mediated endocytosis. Science. 1996;274:2086–2089. [PubMed]
55. Oksvold MP, Skarpen E, Wierod L, Paulsen RE, Huitfeldt HS. Re-localization of activated EGF receptor and its signal transducers to multivesicular compartments downstream of early endosomes in response to EGF. Eur J Cell Biol. 2001;80:285–294. [PubMed]
56. Taub N, Teis D, Ebner HL, Hess MW, Huber LA. Late endosomal traffic of the epidermal growth factor receptor ensures spatial and temporal fidelity of mitogen-activated protein kinase signaling. Mol Biol Cell. 2007;18:4698–4710. [PMC free article] [PubMed]
57. Burke P, Schooler K, Wiley HS. Regulation of epidermal growth factor receptor signaling by endocytosis and intracellular trafficking. Mol Biol Cell. 2001;12:1897–1910. [PMC free article] [PubMed]
58. Jiang X, Sorkin A. Coordinated traffic of Grb2 and Ras during epidermal growth factor receptor endocytosis visualized in living cells. Mol Biol Cell. 2002;13:1522–1535. [PMC free article] [PubMed]
59. Matsuda M, Paterson HF, Rodriguez R, Fensome AC, Ellis MV, Swann K, Katan M. Real time fluorescence imaging of PLC gamma translocation and its interaction with the epidermal growth factor receptor. J Cell Biol. 2001;153:599–612. [PMC free article] [PubMed]
60. Sorkin A, McClure M, Huang F, Carter R. Interaction of EGF receptor and grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy. Curr Biol. 2000;10:1395–1398. [PubMed]
61. Sorkin A, Von Zastrow M. Signal transduction and endocytosis: close encounters of many kinds. Nat Rev Mol Cell Biol. 2002;3:600–614. [PubMed]
62. Wang XJ, Liao HJ, Chattopadhyay A, Carpenter G. EGF-dependent translocation of green fluorescent protein-tagged PLC-gamma1 to the plasma membrane and endosomes. Exp Cell Res. 2001;267:28–36. [PubMed]
63. Pol A, Calvo M, Enrich C. Isolated endosomes from quiescent rat liver contain the signal transduction machinery. Differential distribution of activated Raf-1 and Mek in the endocytic compartment. FEBS Lett. 1998;441:34–38. [PubMed]
64. Roy S, Wyse B, Hancock JF. H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis. Mol Cell Biol. 2002;22:5128–5140. [PMC free article] [PubMed]
65. Omerovic J, Hammond DE, Clague MJ, Prior IA. Ras isoform abundance and signalling in human cancer cell lines. Oncogene. 2008;27:2754–2762. [PMC free article] [PubMed]
66. Zoncu R, Perera RM, Balkin DM, Pirruccello M, Toomre D, De Camilli P. A phosphoinositide switch controls the maturation and signaling properties of APPL endosomes. Cell. 2009;136:1110–1121. [PMC free article] [PubMed]
67. Carpenter G. The EGF receptor: a nexus for trafficking and signaling. Bioessays. 2000;22:697–707. [PubMed]
68. Lu A, Tebar F, Alvarez-Moya B, Lopez-Alcala C, Calvo M, Enrich C, Agell N, Nakamura T, Matsuda M, Bachs O. A clathrin-dependent pathway leads to KRas signaling on late endosomes en route to lysosomes. J Cell Biol. 2009;184:863–879. [PMC free article] [PubMed]
69. Ostenfeld MS, Hoyer-Hansen M, Bastholm L, Fehrenbacher N, Olsen OD, Groth-Pedersen L, Puustinen P, Kirkegaard-Sorensen T, Nylandsted J, Farkas T, et al. Anti-cancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation. Autophagy. 2008;4:487–499. [PubMed]
70. Fehrenbacher N, Bastholm L, Kirkegaard-Sorensen T, Rafn B, Bottzauw T, Nielsen C, Weber E, Shirasawa S, Kallunki T, Jaattela M. Sensitization to the lysosomal cell death pathway by oncogene-induced down-regulation of lysosome-associated membrane proteins 1 and 2. Cancer Res. 2008;68:6623–6633. [PubMed]
71. Jura N, Scotto-Lavino E, Sobczyk A, Bar-Sagi D. Differential modification of Ras proteins by ubiquitination. Mol Cell. 2006;21:679–687. [PubMed]
72. Yan H, Chin ML, Horvath EA, Kane EA, Pfleger CM. Impairment of ubiquitylation by mutation in Drosophila E1 promotes both cell-autonomous and non-cell-autonomous Ras-ERK activation in vivo. J Cell Sci. 2009;122:1461–1470. [PubMed]
73. Galperin E, Sorkin A. Endosomal targeting of MEK2 requires RAF, MEK kinase activity and clathrin-dependent endocytosis. Traffic. 2008;9:1776–1790. [PMC free article] [PubMed]
74. Karandikar M, Cobb MH. Scaffolding and protein interactions in MAP kinase modules. Cell Calcium. 1999;26:219–226. [PubMed]
75. Therrien M, Chang HC, Solomon NM, Karim FD, Wassarman DA, Rubin GM. KSR, a novel protein kinase required for RAS signal transduction. Cell. 1995;83:879–888. [PubMed]
76. Kornfeld K, Hom DB, Horvitz HR. The ksr-1 gene encodes a novel protein kinase involved in Ras-mediated signaling in C. elegans. Cell. 1995;83:903–913. [PubMed]
77. Sundaram M, Han M. The C. elegans ksr-1 gene encodes a novel Raf-related kinase involved in Ras-mediated signal transduction. Cell. 1995;83:889–901. [PubMed]
78. Cacace AM, Michaud NR, Therrien M, Mathes K, Copeland T, Rubin GM, Morrison DK. Identification of constitutive and ras-inducible phosphorylation sites of KSR: implications for 14-3-3 binding, mitogen-activated protein kinase binding, and KSR overexpression. Mol Cell Biol. 1999;19:229–240. [PMC free article] [PubMed]
79. Muller J, Ory S, Copeland T, Piwnica-Worms H, Morrison DK. C-TAK1 regulates Ras signaling by phosphorylating the MAPK scaffold, KSR1. Mol Cell. 2001;8:983–993. [PubMed]
80. Schaeffer HJ, Catling AD, Eblen ST, Collier LS, Krauss A, Weber MJ. MP1: a MEK binding partner that enhances enzymatic activation of the MAP kinase cascade. Science. 1998;281:1668–1671. [PubMed]
81. Teis D, Wunderlich W, Huber LA. Localization of the MP1-MAPK Scaffold Complex to Endosomes Is Mediated by p14 and Required for Signal Transduction. Dev Cell. 2002;3:803–814. [PubMed]
82. Goodman OB, Jr, Krupnick JG, Santini F, Gurevich VV, Penn RB, Gagnon AW, Keen JH, Benovic JL. Beta-arrestin acts as a clathrin adaptor in endocytosis of the beta2-adrenergic receptor. Nature. 1996;383:447–450. [PubMed]
83. Laporte SA, Oakley RH, Zhang J, Holt JA, Ferguson SS, Caron MG, Barak LS. The beta2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis. Proc Natl Acad Sci U S A. 1999;96:3712–3717. [PubMed]
84. Luttrell LM, Roudabush FL, Choy EW, Miller WE, Field ME, Pierce KL, Lefkowitz RJ. Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds. Proc Natl Acad Sci U S A. 2001;98:2449–2454. [PubMed]
85. McDonald PH, Chow CW, Miller WE, Laporte SA, Field ME, Lin FT, Davis RJ, Lefkowitz RJ. Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3. Science. 2000;290:1574–1577. [PubMed]
86. Crosby WH. Blood. 1953;8:769–812. [PubMed]
87. Daaka Y, Luttrell LM, Ahn S, Della Rocca GJ, Ferguson SS, Caron MG, Lefkowitz RJ. Essential role for G protein-coupled receptor endocytosis in the activation of mitogen-activated protein kinase. J Biol Chem. 1998;273:685–688. [PubMed]
88. DeFea KA, Zalevsky J, Thoma MS, Dery O, Mullins RD, Bunnett NW. beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol. 2000;148:1267–1281. [PMC free article] [PubMed]
89. Torii S, Kusakabe M, Yamamoto T, Maekawa M, Nishida E. Sef is a spatial regulator for Ras/MAP kinase signaling. Dev Cell. 2004;7:33–44. [PubMed]
90. Furthauer M, Lin W, Ang SL, Thisse B, Thisse C. Sef is a feedback-induced antagonist of Ras/MAPK-mediated FGF signalling. Nat Cell Biol. 2002;4:170–174. [PubMed]
91. Tsang M, Friesel R, Kudoh T, Dawid IB. Identification of Sef, a novel modulator of FGF signalling. Nat Cell Biol. 2002;4:165–169. [PubMed]
92. Philips MR. Sef: a MEK/ERK catcher on the Golgi. Mol Cell. 2004;15:168–169. [PubMed]