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Ras pathway signaling on endomembranes Trever G Bivona and Mark R Philips Until recently, the plasma membrane has been considered to be a unique platform from which emanate the signaling events regulating or regulated by Ras and its close relatives. For the past few years, the role of endosomes derived from the plasma membrane as platforms for Ras/mitogen-activated protein kinase signaling has been appreciated. More recently, the cytoplasmic face of the Golgi apparatus and endoplasmic reticulum have been shown to host Ras signaling. The biological implications of compartmentalized signaling are only beginning to emerge. Addresses Departments of Medicine, Cell Biology and Pharmacology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA Correspondence: Mark R Philips; e-mail: [email protected]
Current Opinion in Cell Biology 2003, 15:136–142 This review comes from a themed issue on Cell regulation Edited by Pier Paolo di Fiore and Pier Giuseppe Pelicci 0955-0674/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0955-0674(03)00016-4
Abbreviations CAPRI calcium-promoted Ras inactivator CCV clathrin-coated vesicle CFP cyan fluorescent protein EGF epidermal growth factor EGFR epidermal growth factor receptor ERK extracellular-signal-regulated kinase ERI1 ER-associated Ras inhibitor 1 FRET fluorescence resonance energy transfer GAP GTPase-activating protein GEF guanine nucleotide exchange factor GFP green fluorescent protein MAPK mitogen-activated protein kinase MEK MAPK kinase MP1 MEK1 partner 1 NGF nerve growth factor PLCc phospholipase C, g isoform PM plasma membrane PTKR protein tyrosine kinase receptor RasGRP1 Ras guanasyl nucleotide releasing protein 1 RBD Ras-binding domain SH2 Src homology 2 SOS son of sevenless YFP yellow fluorescent protein
Introduction The ability of cells to respond to external stimuli requires that signals are transduced across the plasma membrane (PM) and then through the cytoplasm to reach a variety of Current Opinion in Cell Biology 2003, 15:136–142
organelles, including the cytoskeleton and nucleus. Many early signaling events that follow receptor ligation require a cellular membrane as a platform for the assembly of signaling complexes. Far from inert, the vast endomembrane system is known to host critical signaling pathways including those that regulate the unfolded protein response and cholesterol homeostasis [1,2]. Nevertheless, because the receptors that initiate mitogen-activated protein kinase (MAPK) signaling span the PM and sense extracellular ligands, until recently the endomembrane system has not been considered a platform from which growth signals are generated. Recent advances in cell biology have permitted a series of studies that have challenged this view. The best-characterized pathways that activate Ras/MAPK signaling emanate from protein tyrosine kinase receptors (PTKRs) that span the PM. For example, upon binding of epidermal growth factor (EGF) to its receptor, activation of the receptor’s intrinsic tyrosyl kinase initiates autophosphorylation of tyrosine residues in the carboxy-terminal tail of the receptor, as well as on other proteins, which serve as docking sites for numerous Src homology 2 (SH2)domain-containing proteins. Recruitment of the SH2-containing adaptor Grb2, complexed with the Ras exchange factor SOS, results in activation of Ras followed by recruitment of Raf-1, which initiates a cascade of phosphorylation events involving cytoplasmic MAPKs that ultimately leads to phosphorylation of transcription factors that regulate cell proliferation and differentiation [3]. Ras-family GTPases coordinate a variety of cellular responses to extracellular stimuli and, in doing so, act as critical signal relays. Low-molecular-weight GTPases such as Ras act as molecular switches, cycling between active, GTP-bound, and inactive, GDP-bound, forms. GTP-bound Ras engages effectors (e.g. Raf-1 and phosphatidylinositol 3-kinase [PI3K]). Guanine nucleotide exchange factors (GEFs) stimulate the exchange of GTP for GDP. GTPase-activating proteins (GAPs) augment the intrinsic GTPase activity of Ras and thereby terminate signaling. Thus, Ras activation is modulated by the local balance of GEFs and GAPs. In order for Ras to signal, it must associate with the cytoplasmic leaflet of cellular membranes [4–6]. Ras and related small GTPases (e.g. Rap, R-Ras, M-Ras, Rho, Rac and Cdc42) are synthesized in the cytosol on free polysomes. They contain in their carboxyl termini a hypervariable region that terminates with a CAAX motif that together direct membrane localization. The carboxyterminal CAAX motif is a substrate for three sequential www.current-opinion.com
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post-translational modifications: prenylation, proteolysis and carboxylmethylation. While the prenyltransferase that initiates CAAX processing is cytosolic, the RCE1 protease and prenylcysteine directed carboxylmethyltransferase are localized exclusively in the ER membrane [7,8]. Since CAAX processing is required for membrane association [5], all proteins containing a CAAX motif must, at least transiently, associate with the ER. CAAX processing targets proteins to the ER [6] and adjacent sequences in the hypervariable region direct further trafficking to the PM [5,6]. The hypervariable region contains either a polybasic stretch of amino acids (e.g. K-Ras4B) or cysteine residues (e.g. N- and H-Ras) that are palmitoylated by an acyltransferase that has not been identified at the molecular level but is thought to be associated with the Golgi [9]. Targeting of different Ras proteins to distinct subdomains on the PM [10] or to various intracellular compartments [6,11] may form the basis for specificity in Ras signaling. Because several Ras-family GTPases are localized to internal membranes as well as the PM, recent studies have addressed whether these proteins signal from endomembranes (i.e. the ER, Golgi and vesicular compartments). New applications of imaging technology such as fluorescence resonance energy transfer (FRET) using spectral variants of green fluorescent protein (GFP) in living cells have shed new light on the role of intracellular membranes in Ras signaling. In this review, we will cover recent advances in understanding Ras signaling from platforms other than the PM.
Signaling from endosomes Following activation, PTKRs are internalized from the cell surface via clathrin-dependent and -independent pathways. The most extensively studied of these, EGF receptors (EGFRs), are internalized on clathrin-coated vesicles (CCVs) and sorted either to late endosomes and lysosomes for degradation or to recycling compartments, from which they are returned to the PM [12]. EGFR trafficking has become more complex with the discovery that EGFRs are enriched, along with many other signaling molecules, in caveolae that segregate from clathrincoated pits [12]. Thus, before internalization, activated EGFRs must somehow shuttle out of caveolae and into clathrin-coated pits. Although internalization of EGFRs has been implicated in downregulation of signaling [13], inhibition of clathrin-mediated endocytosis by a dominant-negative form of dynamin revealed that internalization of EGFR was required for MAPK activation [14,15]. Several methods have been used to demonstrate that internalized EGFRs remain activated (i.e. phosphorylated on specific tyrosines). EGFRs at the PM were biotinylated and then followed after internalization with fluorescent streptavidin and fluorescent antibodies to www.current-opinion.com
phosphotyrosine and endosomal markers [16]. Sorkin and co-workers [17] demonstrated internalization of activated, cyan fluorescent protein (CFP)-tagged EGFRs by FRET, using yellow fluorescent protein (YFP)-tagged Grb2 as a phosphotyrosine sensor. Importantly, neither the kinase activity nor the trafficking of EGFR was altered by fusion with GFP [18]. Wouters and Bastiaens [19] made similar observations with EGFR–GFP and microinjected anti-phosphotyrosine antibodies, measuring FRET by fluorescence lifetime imaging microscopy. In addition to activated EGFR, upstream regulators of Ras such as Grb2 and Shc have been localized with EGFR on endosomes [16,20,21]. In one study, Ras was observed to co-localize with Grb2 on vesicles [20], consistent with a report that Ras was present on endosomes isolated from rat liver [22]. Moreover, when the Ras-binding domain (RBD) of Raf-1, which preferentially binds to GTP-bound Ras, was fused to YFP and coexpressed with CFP-tagged H-Ras, FRET was detected on intracellular vesicles. This result indicates that in living cells Ras is activated on vesicles. Some evidence that Ras signaling on endosomes may be isoform-specific comes from recent work by Roy et al. [23], who used a dominant interfering mutant of dynamin to show that H-Ras but not K-Ras signaling was dependent on endocytosis. Despite co-localization of EGFR and associated signaling molecules on endosomes, an unambiguous demonstration of biologically meaningful signaling from endosomes is lacking because endosomal signaling has not been isolated from that ongoing at the PM. Wang et al. [24] have attempted to address this problem by isolating EGFR signaling on endosomes by treating with the EGFR kinase inhibitor AG-1478 and monensin before stimulation with EGF. AG-1478 blocks receptor activation but not internalization and monensin blocks recycling back to the PM. Because the effects of AG-1478 are reversible, washing sets up a condition whereby endosome-associated receptors that remain ligated with EGF become active. Using this method, Wang et al. have shown that EGFR signaling from endosomes leads to cell survival [25]. Like EGFR, neuronal TrkA receptor kinases couple growth factor stimulation with cellular responses such as survival and differentiation. In response to nerve growth factor (NGF), internalized TrkA receptors activate MAPK via Ras. A recent report showed that CCVs contained TrkA and downstream signaling proteins [26]. Phosphorylated TrkA was detected along with Shc, Ras, Raf and MAPK on CCVs isolated from PC12 cells stimulated with NGF. Moreover, increased levels of activated Ras were detected on these CCVs. Ras localized on CCVs was capable of stimulating the phosphorylation of Elk, an endogenous substrate of MAPK. Therefore, similar to epithelial cells and fibroblasts, in PC12 cells Ras signaling is likely to proceed Current Opinion in Cell Biology 2003, 15:136–142
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from intracellular vesicles. Interestingly, both the p85 subunit of PI3K and phospholipase Cg (PLCg) were found associated with CCVs in an NGF-dependent manner suggesting that in PC12 cells multiple pathways downstream of PTKRs may emanate from intracellular vesicles. The molecular machinery that directs internalization of PTKRs might influence endosomal signaling. How endocytic proteins contribute to signaling by PTKRs has been extensively reviewed [27]. Insight into the intimate relationship between PTKR signaling and internalization has come from studies on intersectin, an adaptor protein containing multiple Src homology 3 (SH3) domains that associates with endocytic components, including epsin, dynamin, clathrin, AP2 and Eps15 [28]. Overexpression of intersectin transformed murine fibroblasts and stimulated the c-Jun amino-terminal kinase (JNK) pathway and Elk-1 promoter activation [28]. The growth-stimulatory effects of intersection might be mediated by Ras. Indeed, FRET analysis in living cells revealed that YFPtagged intersectin interacts with CFP–H-Ras on endosomes (R Mahoney, T Bivona, M Philips and J O’Bryan, unpublished data). Because intersectin also interacts with SOS, it may act as a scaffold to promote Ras signaling on endosomes. An attractive hypothesis is that specificity in Ras/MAPK signaling might be achieved through spatial regulation. Accordingly, recent work has focused on understanding how scaffold proteins recruit components of MAPK cascades to specific subcellular compartments. One such scaffold, kinase suppressor of Ras (KSR), binds to MAPK kinase (MEK) and MAPK and was shown to co-localize
with activated Ras at the PM after EGF stimulation [29]. Another scaffold protein, MEK1 partner 1 (MP1) binds MEK1 and facilitates MAPK activation [30]. In response to EGF, MP1 was recruited to late endosomes by the adaptor p14, where it increased phosphorylation of extracellular-signal-regulated kinase (ERK) but not p38. When MP1 was mislocalized from late endosomes by interference with p14 expression, MAPK signaling in response to EGF was attenuated [31]. Interestingly, mislocalization of MP1 impaired the duration but not initial intensity of MAPK activation by EGF. Thus, Ras signaling on late endosomes requires the formation of a specific signaling module that promotes the spatio-temporal regulation of Ras and MAPK activation.
Ras signaling on Golgi and the endoplasmic reticulum Because endosomes are derived from the PM, such that receptors sequestered in this compartment had at one point access to extracellular ligands, continued signaling on the cytoplasmic face of endosomal membranes takes no great leap of imagination. In contrast, signaling on other endomembrane compartments is somewhat counterintuitive. Nevertheless, because post-translational processing of Ras proceeds on ER and because at steady state a significant portion of Ras is localized on Golgi [6], we have investigated whether this intracellular pool of Ras signal in response to growth factors. We used a novel in vivo probe for activated Ras, consisting of the RBD of Raf-1 fused to GFP, to show in living cells that activation of intracellular Ras follows stimulation of PTKRs (Figure 1) [21]. Because signaling of Ras on the ER could only be demonstrated by overexpressing the biosynthetic intermediate
Figure 1
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+ EGF Current Opinion in Cell Biology
Growth factors stimulate GTP/GDP exchange on H-Ras localized at both the PM and Golgi. A COS-1 cell was co-transfected with H-Ras and the RBD of Raf-1 fused to GFP. GFP–RBDRaf-1 serves as an in vivo fluorescent reporter of Ras activation because it binds specifically to GTP-bound Ras. Twenty-four hours after transfection, the cells were serum-starved overnight to minimize Ras activation and then stimulated with EGF. Recruitment of the GFP–RBDRaf-1 reporter from the cytosol to both the PM (white arrows) and Golgi (white arrowhead) indicates that Ras is activated on both compartments. Current Opinion in Cell Biology 2003, 15:136–142
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(unpalmitoylated) H-Ras, the physiological significance of signaling from this compartment remains to be determined. Nevertheless, the rapid (<1 min) activation of unpalmitoyated H-Ras on ER demonstrates a robust mechanism for transmitting PTKR signals across the cytosolic void to Ras on internal membranes. Growth-factorstimulated activation of Ras on Golgi was demonstrated for endogenous Ras using a novel application of FRET designated ‘bystander FRET’ [21]. Thus, Ras signaling from Golgi is likely to be physiologically relevant. Interestingly, the kinetics of Ras activation on PM and Golgi were distinct: activation on PM was rapid (<5 min) and transient, whereas activation on Golgi was delayed (10–20 min) and sustained. This observation suggests that the sustained MAPK signaling previously associated with PC12 cell differentiation [32] may emanate from Golgi. Activation of H-Ras on Golgi but not PM was dependent on Src-family kinases [21]. We have identified a Src/ PLCg/Ca2þ/Ras guanasyl nucleotide releasing protein 1 (RasGRP1)-dependent pathway, distinct from Grb2/SOS, that selectively activates Ras on Golgi (T Bivona and M Philips, unpublished data). This pathway appears to be the predominant one in T cells that express relatively large amounts of RasGRP1. Indeed, N-Ras was activated downstream of the T cell receptor in Jurkat cells exclusively on Golgi (I Perez de Castro, T Bivona, M Philips and A Pellicer, unpublished data). In epithelial cells and fibroblasts where Ras is activated on the cytoplasmic face of both PM and Golgi, the Ca2þ-activated Ras-GAP CAPRI (calcium-promoted Ras inactivator) was active specifically at the PM, allowing for simultaneous activation of Ras on Golgi and inactivation on the PM mediated by a single intracellular second messenger. In Saccharomyces cerevisiae, a novel inhibitor of Ras signaling, ER-associated Ras inhibitor 1 (ERI1), has been identified and found to be expressed exclusively on ER (D Levin, personal communication). Thus, distinct modes of activation and deactivation regulate Ras on different subcellular compartments. This strategy allows the cell to regulate independently activation of Ras on different subcellular compartments (Figure 2). The various functional roles of Ras signaling on distinct subcellular compartments are yet to be determined. At the current level of analysis, the outcome of Ras/MAPK signaling from endosomes does not differ from that previously recognized for PM-associated Ras [25]. Similarly, Ras activation on Golgi or ER is capable of transforming murine fibroblasts [21], suggesting that effector pathways leading to cellular transformation can be engaged from both the PM and intracellular membranes. However, this result does not rule out the possibility that the quality, quantity or balance of signal output might differ from various subcellular compartments in a biologically meaningful way. Some insight has come from studies in which Ras was artificially restricted to ER or Golgi by www.current-opinion.com
fusion with the cytoplasmic tails of transmembrane proteins resident in these compartments. Although there was overlap in the activation of various signaling pathways (ERK versus Akt versus JNK), the relative strengths of the signal outputs depended on sites of activation [21].
Endomembrane signaling by Ras-related GTPases The Rab and Arf families of monomeric GTPases are associated with and regulate the traffic of intracellular membranes, but their roles in transducing signals from PM receptors are largely undefined. In contrast, several GTPases other than Ras known to respond to transmembrane signaling have been localized to internal membranes. These include Rap1, R-Ras, Cdc42, RhoB, RhoD, Rac2 and TC10 [11,33]. Like Ras, these proteins contain CAAX motifs that, in conjunction with upstream sequences, target them to various membrane compartments [11]. Rap1a is a member of the subset of monomeric GTPases that are most closely related to Ras itself. Rap1 was originally identified on the basis of its ability to reverse K-Ras-mediated transformation of murine fibroblasts by competing for Ras effectors [34]. However, recent evidence indicates that Rap1 functions independently from Ras in the regulation of integrin-mediated adhesion [35]. Rap1a, whose hypervariable region is most similar to that of Rac1, has been localized in cultured cells to the PM and intracellular vesicles [36,37]. Using a chimeric FRET probe, Raichu–Rap, to monitor in living cells the sites of Rap1 activation, a recent report showed that Rap1 was activated in response to growth factors primarily on intracellular vesicles [38]. However, Raichu–Rap was targeted to membranes with the hypervariable region of K-Ras4B that directs proteins exclusively to the PM [5,6], raising the question of how vesicular signaling was detected. One explanation is that the relevant Rap1 GEFs might be highly expressed on vesicles along with enough Raichu–Rap to support FRET. Indeed, the Rap GEF RA-GEF-1 has been localized to internal membranes [37]. By contrast, a GFP-tagged form of the Rap GEF RA-GEF-2 was localized to PM in response to M-Ras activation [36], suggesting that activation of Rap1 may also occur on PM. We recently used a novel in vivo probe specific for activated Rap1, consisting of the RBD of RalGDS fused to GFP, to show in living cells that, although the bulk of Rap1 is expressed on endosomes, Rap1 activation by mitogens, M-Ras or T cell receptors elicited activation only on PM (T Bivona and M Philips, unpublished data). Activation of Rap1 on the PM by PTKRs might occur through RA-GEF2, which is activated by M-Ras. Interestingly, M-Ras is activated by the Ras GEF SOS [39] that is recruited to the PM following PTKR stimulation. Thus, Rap1 may be activated on both PM and endomembrane through the Current Opinion in Cell Biology 2003, 15:136–142
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Figure 2
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Compartment-specific regulation of Ras. Dimerization of PTKRs at the cell surface activates Ras on the PM through recruitment of Grb2/SOS complexes. Kinase suppressor of Ras (KSR) serves as a scaffold for Ras signaling on this compartment. Activated receptors interact with Grb2/SOS and Ras on PM-derived endosomes. In this compartment, intersectin (ITSN) serves as a scaffold, binding to SOS and Ras. Ras-independent ERK activation also takes place on the cytoplasmic face of endosomes because the MEK1 scaffold MP1 is recruited to this compartment by the p14 adaptor protein. In addition to Grb2/SOS, PLCg and Src are also recruited to activated PTKRs at the PM. Src participates in the activation of PLCg. Activated PLCg generates the dual second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP30 ), which elevates intracellular Ca2þ. The Ras GEF RasGRP1 responds to DAG and Ca2þ by translocating to the Golgi, where it activates H-Ras. Elevated Ca2þ also activates the Ras GAP CAPRI, directing it to the PM, where it deactivates Ras. Thus, Ca2þ concomitantly activates Ras on one compartment and deactivates it on another. In yeast, ERI1 is expressed on the ER, where it suppresses Ras activation.
action of distinct GEFs with differential affinity for each membrane compartment.
Conclusions: back to the future
now be examined in real time in living cells. These techniques have already led to the appreciation of a role for the endomembrane as a signaling platform for several GTPases.
Understanding how interactions between receptors and intracellular signaling molecules such as adaptors, GTPases and kinases are regulated in intact cells will undoubtedly provide insight into the ways that cells sense and adapt to environmental cues. Recent advances in cell biology have ushered in a new age for the study of old signaling pathways, whose spatio-temporal dynamics can
Elucidating the biological roles for PTKR-induced endomembrane signaling will require multiple methodologies, including validation in transgenic animals. The endomembrane system constitutes the most extensive membrane platform present in eukaryotic cells. Moreover, compartmentalization of signaling events allows for a
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greater diversity of signals than can be generated from a limited number of signaling molecules functioning in only one location. An understanding of the where and when of signaling at the subcellular level, particularly when compartment-specific pathways are involved, might also offer new targets for therapeutics designed to block one but not all outputs from a particular signaling molecule.
Acknowledgements We thank D Levin (Johns Hopkins School of Medicine) and John O’Bryan (National Institute of Environmental Health Science) for sharing unpublished data. We also thank the members of the Philips Lab for insightful discussions. Original work in the authors’ laboratory was supported by National Institutes of Health grants AI36224 and GM55279, by the New York State Breast Cancer Research fellowship, by the Burroughs Wellcome Fund and by a General Clinical Research Center grant from the National Institutes of Health, National Center for Research Resources (M01RR00096), awarded to the New York University School of Medicine.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
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show that activation of EGFRs specifically on endosomes promotes cell survival via phosphatidylinositol 3-kinase. 26. Howe CL, Valletta JS, Rusnak AS, Mobley WC: NGF signaling from clathrin-coated vesicles: evidence that signaling endosomes serve as a platform for the Ras-MAPK pathway. Neuron 2001, 32:801-814. This study identifies in nerve growth factor (NGF)-treated neuronal cells ‘signaling endosomes’ that contain TrkA receptors bound to NGF and associated elements of the Ras/MAPK pathway, including Ras. Interestingly, this compartment could be isolated and shown to phosphorylate Elk-1 in vitro, suggesting that the signaling complexes on endosomes are stable and may account for the transmission of signals over the great distances required in neurons. 27. Di Fiore PP, De Camilli P: Endocytosis and signaling. an inseparable partnership. Cell 2001, 106:1-4. 28. Adams A, Thorn JM, Yamabhai M, Kay BK, O’Bryan JP: Intersectin, an adaptor protein involved in clathrin-mediated endocytosis, activates mitogenic signaling pathways. J Biol Chem 2000, 275:27414-27420. 29. 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. 30. Wunderlich W, Fialka I, Teis D, Alpi A, Pfeifer A, Parton RG, Lottspeich F, Huber LA: A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment. J Cell Biol 2001, 152:765-776. 31. 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. This study showed that mitogen-activated protein kinase (MAPK) activation on endosomes was regulated by the scaffold MP1, localized to this compartment via the adaptor p14. Mislocalization of MP1 via RNA interference of p14 expression attenuated the duration but not initial intensity of MAPK signaling. Compartmentalized signaling on endosomes via MP1 contributes to the spatio-temporal regulation of MAPK activation. 32. Marshall CJ: Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 1995, 80:179-185.
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33. Ehrhardt A, Ehrhardt GR, Guo X, Schrader JW: Ras and relatives– job sharing and networking keep an old family together. Exp Hematol 2002, 30:1089-1106. This is a comprehensive review of the structure and function of Ras and related GTPases. 34. Kitayama H, Sugimoto Y, Matsuzaki T, Ikawa Y, Noda M: A ras-related gene with transformation suppressor activity. Cell 1990, 56:77-84. 35. Reedquist KA, Ross E, Koop EA, Wolthuis RM, Zwartkruis FJ, van Kooyk Y, Salmon M, Buckley CD, Bos JL: The small GTPase, Rap1, mediates CD31-induced integrin adhesion. J Cell Biol 2000, 148:1151-1158. 36. Gao X, Satoh T, Liao Y, Song C, Hu CD, Kariya Ki K, Kataoka T: Identification and characterization of RA-GEF-2, a Rap guanine nucleotide exchange factor that serves as a downstream target of M-Ras. J Biol Chem 2001, 276:42219-42225. 37. Liao Y, Satoh T, Gao X, Jin TG, Hu CD, Kataoka T: RA-GEF-1, a guanine nucleotide exchange factor for Rap1, is activated by translocation induced by association with Rap1*GTP and enhances Rap1-dependent B-Raf activation. J Biol Chem 2001, 276:28478-28483. 38. Mochizuki N, Yamashita S, Kurokawa K, Ohba Y, Nagai T, Miyawaki A, Matsuda M: Spatio-temporal images of growth-factorinduced activation of Ras and Rap1. Nature 2001, 411:1065-1068. These authors developed a series of innovative FRET probes for small GTPases designated ‘Raichu’, and used two of them in this study to show that whereas Ras was activated at the plasma membrane, the closely related small GTPase Rap1a was activated on an intracellular compartment upon stimulation of COS-1 cells with epidermal growth factor. However, the membrane targeting sequence of both Raichu-HRas and Raichu-Rap1 consisted of the hypervariable region of K-Ras4B that targets proteins to the plasma membrane, raising a question as to how the two probes could report activity on distinct subcellular compartments. 39. Quilliam LA, Castro AF, Rogers-Graham KS, Martin CB, Der CJ, Bi C: M-Ras/R-Ras3, a transforming ras protein regulated by Sos1, GRF1, and p120 Ras GTPase-activating protein, interacts with the putative Ras effector AF6. J Biol Chem 1999, 274:23850-23857.
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Ras pathway signaling on endomembranes Trever G Bivona and Mark R Philips Until recently, the plasma membrane has been considered to be a unique platform from which emanate the signaling events regulating or regulated by Ras and its close relatives. For the past few years, the role of endosomes derived from the plasma membrane as platforms for Ras/mitogen-activated protein kinase signaling has been appreciated. More recently, the cytoplasmic face of the Golgi apparatus and endoplasmic reticulum have been shown to host Ras signaling. The biological implications of compartmentalized signaling are only beginning to emerge. Addresses Departments of Medicine, Cell Biology and Pharmacology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA Correspondence: Mark R Philips; e-mail: [email protected]
Current Opinion in Cell Biology 2003, 15:136–142 This review comes from a themed issue on Cell regulation Edited by Pier Paolo di Fiore and Pier Giuseppe Pelicci 0955-0674/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0955-0674(03)00016-4
Abbreviations CAPRI calcium-promoted Ras inactivator CCV clathrin-coated vesicle CFP cyan fluorescent protein EGF epidermal growth factor EGFR epidermal growth factor receptor ERK extracellular-signal-regulated kinase ERI1 ER-associated Ras inhibitor 1 FRET fluorescence resonance energy transfer GAP GTPase-activating protein GEF guanine nucleotide exchange factor GFP green fluorescent protein MAPK mitogen-activated protein kinase MEK MAPK kinase MP1 MEK1 partner 1 NGF nerve growth factor PLCc phospholipase C, g isoform PM plasma membrane PTKR protein tyrosine kinase receptor RasGRP1 Ras guanasyl nucleotide releasing protein 1 RBD Ras-binding domain SH2 Src homology 2 SOS son of sevenless YFP yellow fluorescent protein
Introduction The ability of cells to respond to external stimuli requires that signals are transduced across the plasma membrane (PM) and then through the cytoplasm to reach a variety of Current Opinion in Cell Biology 2003, 15:136–142
organelles, including the cytoskeleton and nucleus. Many early signaling events that follow receptor ligation require a cellular membrane as a platform for the assembly of signaling complexes. Far from inert, the vast endomembrane system is known to host critical signaling pathways including those that regulate the unfolded protein response and cholesterol homeostasis [1,2]. Nevertheless, because the receptors that initiate mitogen-activated protein kinase (MAPK) signaling span the PM and sense extracellular ligands, until recently the endomembrane system has not been considered a platform from which growth signals are generated. Recent advances in cell biology have permitted a series of studies that have challenged this view. The best-characterized pathways that activate Ras/MAPK signaling emanate from protein tyrosine kinase receptors (PTKRs) that span the PM. For example, upon binding of epidermal growth factor (EGF) to its receptor, activation of the receptor’s intrinsic tyrosyl kinase initiates autophosphorylation of tyrosine residues in the carboxy-terminal tail of the receptor, as well as on other proteins, which serve as docking sites for numerous Src homology 2 (SH2)domain-containing proteins. Recruitment of the SH2-containing adaptor Grb2, complexed with the Ras exchange factor SOS, results in activation of Ras followed by recruitment of Raf-1, which initiates a cascade of phosphorylation events involving cytoplasmic MAPKs that ultimately leads to phosphorylation of transcription factors that regulate cell proliferation and differentiation [3]. Ras-family GTPases coordinate a variety of cellular responses to extracellular stimuli and, in doing so, act as critical signal relays. Low-molecular-weight GTPases such as Ras act as molecular switches, cycling between active, GTP-bound, and inactive, GDP-bound, forms. GTP-bound Ras engages effectors (e.g. Raf-1 and phosphatidylinositol 3-kinase [PI3K]). Guanine nucleotide exchange factors (GEFs) stimulate the exchange of GTP for GDP. GTPase-activating proteins (GAPs) augment the intrinsic GTPase activity of Ras and thereby terminate signaling. Thus, Ras activation is modulated by the local balance of GEFs and GAPs. In order for Ras to signal, it must associate with the cytoplasmic leaflet of cellular membranes [4–6]. Ras and related small GTPases (e.g. Rap, R-Ras, M-Ras, Rho, Rac and Cdc42) are synthesized in the cytosol on free polysomes. They contain in their carboxyl termini a hypervariable region that terminates with a CAAX motif that together direct membrane localization. The carboxyterminal CAAX motif is a substrate for three sequential www.current-opinion.com
Ras pathway signaling on endomembranes Bivona and Philips 137
post-translational modifications: prenylation, proteolysis and carboxylmethylation. While the prenyltransferase that initiates CAAX processing is cytosolic, the RCE1 protease and prenylcysteine directed carboxylmethyltransferase are localized exclusively in the ER membrane [7,8]. Since CAAX processing is required for membrane association [5], all proteins containing a CAAX motif must, at least transiently, associate with the ER. CAAX processing targets proteins to the ER [6] and adjacent sequences in the hypervariable region direct further trafficking to the PM [5,6]. The hypervariable region contains either a polybasic stretch of amino acids (e.g. K-Ras4B) or cysteine residues (e.g. N- and H-Ras) that are palmitoylated by an acyltransferase that has not been identified at the molecular level but is thought to be associated with the Golgi [9]. Targeting of different Ras proteins to distinct subdomains on the PM [10] or to various intracellular compartments [6,11] may form the basis for specificity in Ras signaling. Because several Ras-family GTPases are localized to internal membranes as well as the PM, recent studies have addressed whether these proteins signal from endomembranes (i.e. the ER, Golgi and vesicular compartments). New applications of imaging technology such as fluorescence resonance energy transfer (FRET) using spectral variants of green fluorescent protein (GFP) in living cells have shed new light on the role of intracellular membranes in Ras signaling. In this review, we will cover recent advances in understanding Ras signaling from platforms other than the PM.
Signaling from endosomes Following activation, PTKRs are internalized from the cell surface via clathrin-dependent and -independent pathways. The most extensively studied of these, EGF receptors (EGFRs), are internalized on clathrin-coated vesicles (CCVs) and sorted either to late endosomes and lysosomes for degradation or to recycling compartments, from which they are returned to the PM [12]. EGFR trafficking has become more complex with the discovery that EGFRs are enriched, along with many other signaling molecules, in caveolae that segregate from clathrincoated pits [12]. Thus, before internalization, activated EGFRs must somehow shuttle out of caveolae and into clathrin-coated pits. Although internalization of EGFRs has been implicated in downregulation of signaling [13], inhibition of clathrin-mediated endocytosis by a dominant-negative form of dynamin revealed that internalization of EGFR was required for MAPK activation [14,15]. Several methods have been used to demonstrate that internalized EGFRs remain activated (i.e. phosphorylated on specific tyrosines). EGFRs at the PM were biotinylated and then followed after internalization with fluorescent streptavidin and fluorescent antibodies to www.current-opinion.com
phosphotyrosine and endosomal markers [16]. Sorkin and co-workers [17] demonstrated internalization of activated, cyan fluorescent protein (CFP)-tagged EGFRs by FRET, using yellow fluorescent protein (YFP)-tagged Grb2 as a phosphotyrosine sensor. Importantly, neither the kinase activity nor the trafficking of EGFR was altered by fusion with GFP [18]. Wouters and Bastiaens [19] made similar observations with EGFR–GFP and microinjected anti-phosphotyrosine antibodies, measuring FRET by fluorescence lifetime imaging microscopy. In addition to activated EGFR, upstream regulators of Ras such as Grb2 and Shc have been localized with EGFR on endosomes [16,20,21]. In one study, Ras was observed to co-localize with Grb2 on vesicles [20], consistent with a report that Ras was present on endosomes isolated from rat liver [22]. Moreover, when the Ras-binding domain (RBD) of Raf-1, which preferentially binds to GTP-bound Ras, was fused to YFP and coexpressed with CFP-tagged H-Ras, FRET was detected on intracellular vesicles. This result indicates that in living cells Ras is activated on vesicles. Some evidence that Ras signaling on endosomes may be isoform-specific comes from recent work by Roy et al. [23], who used a dominant interfering mutant of dynamin to show that H-Ras but not K-Ras signaling was dependent on endocytosis. Despite co-localization of EGFR and associated signaling molecules on endosomes, an unambiguous demonstration of biologically meaningful signaling from endosomes is lacking because endosomal signaling has not been isolated from that ongoing at the PM. Wang et al. [24] have attempted to address this problem by isolating EGFR signaling on endosomes by treating with the EGFR kinase inhibitor AG-1478 and monensin before stimulation with EGF. AG-1478 blocks receptor activation but not internalization and monensin blocks recycling back to the PM. Because the effects of AG-1478 are reversible, washing sets up a condition whereby endosome-associated receptors that remain ligated with EGF become active. Using this method, Wang et al. have shown that EGFR signaling from endosomes leads to cell survival [25]. Like EGFR, neuronal TrkA receptor kinases couple growth factor stimulation with cellular responses such as survival and differentiation. In response to nerve growth factor (NGF), internalized TrkA receptors activate MAPK via Ras. A recent report showed that CCVs contained TrkA and downstream signaling proteins [26]. Phosphorylated TrkA was detected along with Shc, Ras, Raf and MAPK on CCVs isolated from PC12 cells stimulated with NGF. Moreover, increased levels of activated Ras were detected on these CCVs. Ras localized on CCVs was capable of stimulating the phosphorylation of Elk, an endogenous substrate of MAPK. Therefore, similar to epithelial cells and fibroblasts, in PC12 cells Ras signaling is likely to proceed Current Opinion in Cell Biology 2003, 15:136–142
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from intracellular vesicles. Interestingly, both the p85 subunit of PI3K and phospholipase Cg (PLCg) were found associated with CCVs in an NGF-dependent manner suggesting that in PC12 cells multiple pathways downstream of PTKRs may emanate from intracellular vesicles. The molecular machinery that directs internalization of PTKRs might influence endosomal signaling. How endocytic proteins contribute to signaling by PTKRs has been extensively reviewed [27]. Insight into the intimate relationship between PTKR signaling and internalization has come from studies on intersectin, an adaptor protein containing multiple Src homology 3 (SH3) domains that associates with endocytic components, including epsin, dynamin, clathrin, AP2 and Eps15 [28]. Overexpression of intersectin transformed murine fibroblasts and stimulated the c-Jun amino-terminal kinase (JNK) pathway and Elk-1 promoter activation [28]. The growth-stimulatory effects of intersection might be mediated by Ras. Indeed, FRET analysis in living cells revealed that YFPtagged intersectin interacts with CFP–H-Ras on endosomes (R Mahoney, T Bivona, M Philips and J O’Bryan, unpublished data). Because intersectin also interacts with SOS, it may act as a scaffold to promote Ras signaling on endosomes. An attractive hypothesis is that specificity in Ras/MAPK signaling might be achieved through spatial regulation. Accordingly, recent work has focused on understanding how scaffold proteins recruit components of MAPK cascades to specific subcellular compartments. One such scaffold, kinase suppressor of Ras (KSR), binds to MAPK kinase (MEK) and MAPK and was shown to co-localize
with activated Ras at the PM after EGF stimulation [29]. Another scaffold protein, MEK1 partner 1 (MP1) binds MEK1 and facilitates MAPK activation [30]. In response to EGF, MP1 was recruited to late endosomes by the adaptor p14, where it increased phosphorylation of extracellular-signal-regulated kinase (ERK) but not p38. When MP1 was mislocalized from late endosomes by interference with p14 expression, MAPK signaling in response to EGF was attenuated [31]. Interestingly, mislocalization of MP1 impaired the duration but not initial intensity of MAPK activation by EGF. Thus, Ras signaling on late endosomes requires the formation of a specific signaling module that promotes the spatio-temporal regulation of Ras and MAPK activation.
Ras signaling on Golgi and the endoplasmic reticulum Because endosomes are derived from the PM, such that receptors sequestered in this compartment had at one point access to extracellular ligands, continued signaling on the cytoplasmic face of endosomal membranes takes no great leap of imagination. In contrast, signaling on other endomembrane compartments is somewhat counterintuitive. Nevertheless, because post-translational processing of Ras proceeds on ER and because at steady state a significant portion of Ras is localized on Golgi [6], we have investigated whether this intracellular pool of Ras signal in response to growth factors. We used a novel in vivo probe for activated Ras, consisting of the RBD of Raf-1 fused to GFP, to show in living cells that activation of intracellular Ras follows stimulation of PTKRs (Figure 1) [21]. Because signaling of Ras on the ER could only be demonstrated by overexpressing the biosynthetic intermediate
Figure 1
– EGF
+ EGF Current Opinion in Cell Biology
Growth factors stimulate GTP/GDP exchange on H-Ras localized at both the PM and Golgi. A COS-1 cell was co-transfected with H-Ras and the RBD of Raf-1 fused to GFP. GFP–RBDRaf-1 serves as an in vivo fluorescent reporter of Ras activation because it binds specifically to GTP-bound Ras. Twenty-four hours after transfection, the cells were serum-starved overnight to minimize Ras activation and then stimulated with EGF. Recruitment of the GFP–RBDRaf-1 reporter from the cytosol to both the PM (white arrows) and Golgi (white arrowhead) indicates that Ras is activated on both compartments. Current Opinion in Cell Biology 2003, 15:136–142
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(unpalmitoylated) H-Ras, the physiological significance of signaling from this compartment remains to be determined. Nevertheless, the rapid (<1 min) activation of unpalmitoyated H-Ras on ER demonstrates a robust mechanism for transmitting PTKR signals across the cytosolic void to Ras on internal membranes. Growth-factorstimulated activation of Ras on Golgi was demonstrated for endogenous Ras using a novel application of FRET designated ‘bystander FRET’ [21]. Thus, Ras signaling from Golgi is likely to be physiologically relevant. Interestingly, the kinetics of Ras activation on PM and Golgi were distinct: activation on PM was rapid (<5 min) and transient, whereas activation on Golgi was delayed (10–20 min) and sustained. This observation suggests that the sustained MAPK signaling previously associated with PC12 cell differentiation [32] may emanate from Golgi. Activation of H-Ras on Golgi but not PM was dependent on Src-family kinases [21]. We have identified a Src/ PLCg/Ca2þ/Ras guanasyl nucleotide releasing protein 1 (RasGRP1)-dependent pathway, distinct from Grb2/SOS, that selectively activates Ras on Golgi (T Bivona and M Philips, unpublished data). This pathway appears to be the predominant one in T cells that express relatively large amounts of RasGRP1. Indeed, N-Ras was activated downstream of the T cell receptor in Jurkat cells exclusively on Golgi (I Perez de Castro, T Bivona, M Philips and A Pellicer, unpublished data). In epithelial cells and fibroblasts where Ras is activated on the cytoplasmic face of both PM and Golgi, the Ca2þ-activated Ras-GAP CAPRI (calcium-promoted Ras inactivator) was active specifically at the PM, allowing for simultaneous activation of Ras on Golgi and inactivation on the PM mediated by a single intracellular second messenger. In Saccharomyces cerevisiae, a novel inhibitor of Ras signaling, ER-associated Ras inhibitor 1 (ERI1), has been identified and found to be expressed exclusively on ER (D Levin, personal communication). Thus, distinct modes of activation and deactivation regulate Ras on different subcellular compartments. This strategy allows the cell to regulate independently activation of Ras on different subcellular compartments (Figure 2). The various functional roles of Ras signaling on distinct subcellular compartments are yet to be determined. At the current level of analysis, the outcome of Ras/MAPK signaling from endosomes does not differ from that previously recognized for PM-associated Ras [25]. Similarly, Ras activation on Golgi or ER is capable of transforming murine fibroblasts [21], suggesting that effector pathways leading to cellular transformation can be engaged from both the PM and intracellular membranes. However, this result does not rule out the possibility that the quality, quantity or balance of signal output might differ from various subcellular compartments in a biologically meaningful way. Some insight has come from studies in which Ras was artificially restricted to ER or Golgi by www.current-opinion.com
fusion with the cytoplasmic tails of transmembrane proteins resident in these compartments. Although there was overlap in the activation of various signaling pathways (ERK versus Akt versus JNK), the relative strengths of the signal outputs depended on sites of activation [21].
Endomembrane signaling by Ras-related GTPases The Rab and Arf families of monomeric GTPases are associated with and regulate the traffic of intracellular membranes, but their roles in transducing signals from PM receptors are largely undefined. In contrast, several GTPases other than Ras known to respond to transmembrane signaling have been localized to internal membranes. These include Rap1, R-Ras, Cdc42, RhoB, RhoD, Rac2 and TC10 [11,33]. Like Ras, these proteins contain CAAX motifs that, in conjunction with upstream sequences, target them to various membrane compartments [11]. Rap1a is a member of the subset of monomeric GTPases that are most closely related to Ras itself. Rap1 was originally identified on the basis of its ability to reverse K-Ras-mediated transformation of murine fibroblasts by competing for Ras effectors [34]. However, recent evidence indicates that Rap1 functions independently from Ras in the regulation of integrin-mediated adhesion [35]. Rap1a, whose hypervariable region is most similar to that of Rac1, has been localized in cultured cells to the PM and intracellular vesicles [36,37]. Using a chimeric FRET probe, Raichu–Rap, to monitor in living cells the sites of Rap1 activation, a recent report showed that Rap1 was activated in response to growth factors primarily on intracellular vesicles [38]. However, Raichu–Rap was targeted to membranes with the hypervariable region of K-Ras4B that directs proteins exclusively to the PM [5,6], raising the question of how vesicular signaling was detected. One explanation is that the relevant Rap1 GEFs might be highly expressed on vesicles along with enough Raichu–Rap to support FRET. Indeed, the Rap GEF RA-GEF-1 has been localized to internal membranes [37]. By contrast, a GFP-tagged form of the Rap GEF RA-GEF-2 was localized to PM in response to M-Ras activation [36], suggesting that activation of Rap1 may also occur on PM. We recently used a novel in vivo probe specific for activated Rap1, consisting of the RBD of RalGDS fused to GFP, to show in living cells that, although the bulk of Rap1 is expressed on endosomes, Rap1 activation by mitogens, M-Ras or T cell receptors elicited activation only on PM (T Bivona and M Philips, unpublished data). Activation of Rap1 on the PM by PTKRs might occur through RA-GEF2, which is activated by M-Ras. Interestingly, M-Ras is activated by the Ras GEF SOS [39] that is recruited to the PM following PTKR stimulation. Thus, Rap1 may be activated on both PM and endomembrane through the Current Opinion in Cell Biology 2003, 15:136–142
140 Cell regulation
Figure 2
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s Ra ITSN
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Compartment-specific regulation of Ras. Dimerization of PTKRs at the cell surface activates Ras on the PM through recruitment of Grb2/SOS complexes. Kinase suppressor of Ras (KSR) serves as a scaffold for Ras signaling on this compartment. Activated receptors interact with Grb2/SOS and Ras on PM-derived endosomes. In this compartment, intersectin (ITSN) serves as a scaffold, binding to SOS and Ras. Ras-independent ERK activation also takes place on the cytoplasmic face of endosomes because the MEK1 scaffold MP1 is recruited to this compartment by the p14 adaptor protein. In addition to Grb2/SOS, PLCg and Src are also recruited to activated PTKRs at the PM. Src participates in the activation of PLCg. Activated PLCg generates the dual second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP30 ), which elevates intracellular Ca2þ. The Ras GEF RasGRP1 responds to DAG and Ca2þ by translocating to the Golgi, where it activates H-Ras. Elevated Ca2þ also activates the Ras GAP CAPRI, directing it to the PM, where it deactivates Ras. Thus, Ca2þ concomitantly activates Ras on one compartment and deactivates it on another. In yeast, ERI1 is expressed on the ER, where it suppresses Ras activation.
action of distinct GEFs with differential affinity for each membrane compartment.
Conclusions: back to the future
now be examined in real time in living cells. These techniques have already led to the appreciation of a role for the endomembrane as a signaling platform for several GTPases.
Understanding how interactions between receptors and intracellular signaling molecules such as adaptors, GTPases and kinases are regulated in intact cells will undoubtedly provide insight into the ways that cells sense and adapt to environmental cues. Recent advances in cell biology have ushered in a new age for the study of old signaling pathways, whose spatio-temporal dynamics can
Elucidating the biological roles for PTKR-induced endomembrane signaling will require multiple methodologies, including validation in transgenic animals. The endomembrane system constitutes the most extensive membrane platform present in eukaryotic cells. Moreover, compartmentalization of signaling events allows for a
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greater diversity of signals than can be generated from a limited number of signaling molecules functioning in only one location. An understanding of the where and when of signaling at the subcellular level, particularly when compartment-specific pathways are involved, might also offer new targets for therapeutics designed to block one but not all outputs from a particular signaling molecule.
Acknowledgements We thank D Levin (Johns Hopkins School of Medicine) and John O’Bryan (National Institute of Environmental Health Science) for sharing unpublished data. We also thank the members of the Philips Lab for insightful discussions. Original work in the authors’ laboratory was supported by National Institutes of Health grants AI36224 and GM55279, by the New York State Breast Cancer Research fellowship, by the Burroughs Wellcome Fund and by a General Clinical Research Center grant from the National Institutes of Health, National Center for Research Resources (M01RR00096), awarded to the New York University School of Medicine.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
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24. Wang Y, Pennock S, Chen X, Wang Z: Internalization of inactive EGF receptor into endosomes and the subsequent activation of endosome-associated EGF receptors. Sci STKE 2002, 2002:PL17. 25. Wang Y, Pennock S, Chen X, Wang Z: Endosomal signaling of epidermal growth factor receptor stimulates signal transduction pathways leading to cell survival. Mol Cell Biol 2002, 22:7279-7290. Studies on the specific role(s) of epidermal growth factor receptor (EGFR) signaling from endosomes has been confounded by the inability to activate receptors on this compartment without first provoking activation on plasma membrane. This study defined experimental conditions whereby signaling by EGFRs within endosomes was distinguished from EGFRs within the plasma membrane. The authors used this method to Current Opinion in Cell Biology 2003, 15:136–142
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show that activation of EGFRs specifically on endosomes promotes cell survival via phosphatidylinositol 3-kinase. 26. Howe CL, Valletta JS, Rusnak AS, Mobley WC: NGF signaling from clathrin-coated vesicles: evidence that signaling endosomes serve as a platform for the Ras-MAPK pathway. Neuron 2001, 32:801-814. This study identifies in nerve growth factor (NGF)-treated neuronal cells ‘signaling endosomes’ that contain TrkA receptors bound to NGF and associated elements of the Ras/MAPK pathway, including Ras. Interestingly, this compartment could be isolated and shown to phosphorylate Elk-1 in vitro, suggesting that the signaling complexes on endosomes are stable and may account for the transmission of signals over the great distances required in neurons. 27. Di Fiore PP, De Camilli P: Endocytosis and signaling. an inseparable partnership. Cell 2001, 106:1-4. 28. Adams A, Thorn JM, Yamabhai M, Kay BK, O’Bryan JP: Intersectin, an adaptor protein involved in clathrin-mediated endocytosis, activates mitogenic signaling pathways. J Biol Chem 2000, 275:27414-27420. 29. 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. 30. Wunderlich W, Fialka I, Teis D, Alpi A, Pfeifer A, Parton RG, Lottspeich F, Huber LA: A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment. J Cell Biol 2001, 152:765-776. 31. 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. This study showed that mitogen-activated protein kinase (MAPK) activation on endosomes was regulated by the scaffold MP1, localized to this compartment via the adaptor p14. Mislocalization of MP1 via RNA interference of p14 expression attenuated the duration but not initial intensity of MAPK signaling. Compartmentalized signaling on endosomes via MP1 contributes to the spatio-temporal regulation of MAPK activation. 32. Marshall CJ: Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 1995, 80:179-185.
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33. Ehrhardt A, Ehrhardt GR, Guo X, Schrader JW: Ras and relatives– job sharing and networking keep an old family together. Exp Hematol 2002, 30:1089-1106. This is a comprehensive review of the structure and function of Ras and related GTPases. 34. Kitayama H, Sugimoto Y, Matsuzaki T, Ikawa Y, Noda M: A ras-related gene with transformation suppressor activity. Cell 1990, 56:77-84. 35. Reedquist KA, Ross E, Koop EA, Wolthuis RM, Zwartkruis FJ, van Kooyk Y, Salmon M, Buckley CD, Bos JL: The small GTPase, Rap1, mediates CD31-induced integrin adhesion. J Cell Biol 2000, 148:1151-1158. 36. Gao X, Satoh T, Liao Y, Song C, Hu CD, Kariya Ki K, Kataoka T: Identification and characterization of RA-GEF-2, a Rap guanine nucleotide exchange factor that serves as a downstream target of M-Ras. J Biol Chem 2001, 276:42219-42225. 37. Liao Y, Satoh T, Gao X, Jin TG, Hu CD, Kataoka T: RA-GEF-1, a guanine nucleotide exchange factor for Rap1, is activated by translocation induced by association with Rap1*GTP and enhances Rap1-dependent B-Raf activation. J Biol Chem 2001, 276:28478-28483. 38. Mochizuki N, Yamashita S, Kurokawa K, Ohba Y, Nagai T, Miyawaki A, Matsuda M: Spatio-temporal images of growth-factorinduced activation of Ras and Rap1. Nature 2001, 411:1065-1068. These authors developed a series of innovative FRET probes for small GTPases designated ‘Raichu’, and used two of them in this study to show that whereas Ras was activated at the plasma membrane, the closely related small GTPase Rap1a was activated on an intracellular compartment upon stimulation of COS-1 cells with epidermal growth factor. However, the membrane targeting sequence of both Raichu-HRas and Raichu-Rap1 consisted of the hypervariable region of K-Ras4B that targets proteins to the plasma membrane, raising a question as to how the two probes could report activity on distinct subcellular compartments. 39. Quilliam LA, Castro AF, Rogers-Graham KS, Martin CB, Der CJ, Bi C: M-Ras/R-Ras3, a transforming ras protein regulated by Sos1, GRF1, and p120 Ras GTPase-activating protein, interacts with the putative Ras effector AF6. J Biol Chem 1999, 274:23850-23857.
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