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Ras signaling on the Golgi
Ras signaling on the Golgi Steven E Quatela and Mark R Philips The discovery that Ras proteins are modified by enzymes restricted to the endoplasmic reticulum and Golgi apparatus and that, at steady state, a significant pool of Ras is localized on the Golgi has led to the hypothesis that Ras can become activated on and signal from intracellular membranes. Fluorescent probes capable of showing when and where in living cells Ras becomes activated together with studies of Ras proteins stringently tethered to intracellular membranes have confirmed this hypothesis. Thus, recent studies of Ras have contributed to the rapidly expanding field of compartmentalized signaling. Addresses Department of Pharmacology, MSB 251, NYU School of Medicine, 550 First Avenue, New York, NY 10016 Corresponding author: Philips, Mark R ([email protected])
Current Opinion in Cell Biology 2006, 18:162–167 This review comes from a themed issue on Cell regulation Edited by Claude Prigent and Bruno Goud Available online 20th February 2006 0955-0674/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2006.02.004
Association of Ras proteins with the cytosolic leaflet of cellular membranes is required for biological activity [4– 6]. Ras proteins are synthesized as hydrophilic proteins on free polysomes in the cytosol and are targeted posttranslationally to cellular membranes by virtue of a series of modifications that include prenylation, proteolysis and carboxyl methylation [7]. These modifications are directed by a conserved C-terminal CAAX sequence where C is cysteine, A is usually an aliphatic amino acid and X is any amino acid [8]. In the case of Ras, the CAAX sequence is recognized by farnesyl transferase (FTase) [9], a cytosolic enzyme that catalyzes the addition of a 15carbon farnesyl isoprenoid to the CAAX cysteine via a stable thioether linkage [10]. Once prenylated, the Sisoprenyl CAAX moiety becomes a substrate for Rce1, an ER-restricted protease that cleaves the AAX sequence [7,11]. The newly C-terminal prenylcysteine is then recognized by a third enzyme, isoprenylcysteine carboxyl methyltransferase (Icmt), also restricted to the ER, that methylesterifies the carboxyl group [7,12]. The end result of these three modifications is to create a hydrophobic domain at the C-terminus that mediates membrane association. N-Ras, H-Ras and Kras4A, but not K-Ras4B, are further modified by the addition of one or two palmitic acids just upstream of the farnesylcysteine. A genetic screen in S. cerevisiae identified Erf2/Erf4 as the Ras palmitoyltransferase [13]. Sequence homology with Erf2/Erf4 revealed the human palmitoyltransferase to be a complex of the proteins DHHC9 and GCP16 [14], both of which localize to the Golgi apparatus [15].
Introduction Ras proteins are small GTPases that regulate cell growth, proliferation, differentiation and apoptosis. Ras genes were first recognized as the transforming principles of two retroviruses and mutated alleles of the cellular protooncogenes have been associated with a variety of human cancer [1]. Oncogenic Ras can induce the growth and transformation of cells both in vitro and in vivo [2]. Ras proteins are founding members of a large superfamily of monomeric GTPases that function as molecular switches, cycling between inactive, GDP-bound and active, GTPbound forms. Guanine nucleotide-exchange factors (GEFs) mediate signal-induced activation of Ras by stimulating the exchange of GDP for GTP; this induces a conformational change in the protein [3] that permits interaction with downstream effectors such as Raf-1, PI3K, and RalGDS. The active state of Ras is self-limited by its intrinsic GTPase activity and this relatively weak activity is greatly accelerated by GTPase activating proteins (GAPs). Mammalian genomes encode three ras genes that give rise to four ubiquitously expressed gene products: N-Ras, H-Ras, K-Ras4A and K-Ras4B. Current Opinion in Cell Biology 2006, 18:162–167
The post-translational modifications to the C-terminal CAAX motif of Ras were originally thought to target nascent cytosolic Ras proteins directly to the plasma membrane (PM). The discovery that the enzymes that further modify prenylated proteins are restricted to the ER or Golgi [12,14–17] suggested that nascent Ras must traffic to the PM via the endomembrane. This prediction was verified by studying the trafficking of green fluorescent protein (GFP)-tagged Ras [5,18]. Once fully modified on the endomembrane, the subsequent transfer of Ras proteins to the PM requires a second signal found in the C-terminal hypervariable regions of Ras proteins immediately adjacent to their CAAX motifs. For N-Ras and H-Ras this signal consists of one or two cysteine residues that serve as sites of palmitoylation. For KRas4B, the signal consists of a polybasic region rich in lysine residues [6,19]. Whereas prenylation alone serves as a relatively weak membrane anchor, palmitoylation markedly increases affinity for membranes, trapping NRas and H-Ras in the phospholipid bilayer. The positively charged polybasic region of K-Ras is thought to www.sciencedirect.com
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associate with the negatively charged head groups of the inner leaflet phospholipids of the PM. Because Ras GTPases link receptors, such as protein tyrosine kinase receptors (PTKRs), that traverse the PM with cytoplasmic effector pathways that regulate cellular function, it is intuitive to think that Ras signaling occurs at the PM. The discovery that Ras proteins visit the endomembrane en route to the PM, and that at steady-state the palmitoylated isoforms of Ras are highly expressed on the Golgi apparatus, led to the hypothesis that Ras signaling may also occur on intracellular compartments. This hypothesis has been tested in living cells with fluorescent probes and shown to be correct: Ras and/ or MAPK signaling originates from the cytoplasmic face of endosomes, the ER, the Golgi apparatus and mitochondria as well as from the PM. In this review, we will cover the most recent advances in understanding Ras signaling from the Golgi apparatus.
Ras activity on the Golgi The observation that activated Ras accumulates on the Golgi was made possible by the advent of genetically encoded fluorescent probes that are capable of reporting where and when Ras becomes loaded with GTP in living cells [20–22]. The first of these was the founding member of the so-called Raichu probes developed by Matsuda and colleagues. Raichu-Ras is a FRET-based probe where Ras and the Ras binding domain (RBD) of Raf-1 are in cis on a single polypeptide that is flanked by CFP and YFP (cyan and yellow fluorescent proteins) such that, when the exogenous Ras is GTP-bound, the molecule assumes a hairpin configuration and a FRET signal is generated [21]. Raichu-Ras is targeted to the PM with the 20-aminoacid C-terminus of K-Ras such that it is spatially biased in
its reporting. Moreover, because the probe incorporates both the GTPase and the cognate RBD, which are therefore overexpressed, rather than reporting local Ras activation it actually reports the local balance of GEFs and GAPs [23]. As expected by its PM targeting, Raichu-Ras did not report Ras activation on the Golgi [21]. Perhaps the most informative probe for GTP-bound Ras is the simplest, consisting of the RBD fused to GFP. This probe reports Ras activation in a spatiotemporal manner by translocating from the cytosol and nucleoplasm to membrane compartments upon which Ras becomes activated (Figure 1). Among the advantages of this probe is that it is not itself targeted in any way, meaning that it is spatially unbiased. The major disadvantage of this probe is that unless a GEF is overexpressed [24], it is not sensitive enough to detect endogenous GTP-bound Ras and therefore requires overexpression of wild-type Ras proteins. Using GFP–RBD, Chiu et al. showed that upon stimulation of fibroblasts with epidermal growth factor (EGF), Ras became activated at both the PM and the Golgi [20]. These observations have now been confirmed by several laboratories [24,25,26]. Interestingly, the kinetics of activation differed between the two compartments: whereas recruitment of GFP–RBD to the PM was rapid and transient (1–3 min onset, 10 min duration), activation of Ras on the Golgi was delayed and sustained (onset 10– 20 min, duration >60 min). The kinetics and relative extent of compartment-specific activation is cell-typespecific. In Jurkat T cells stimulated through the antigen receptor (TCR), activation on the Golgi was rapid (1– 3 min) and, surprisingly, no activation on the PM was observed [27]. Ras activation exclusively on the Golgi downstream of TCR signaling was also observed in primary T cells, as was constitutive activation of Golgi-
Figure 1
Detection of endogenous Ras activation on the Golgi of Jurkat T cells using bystander FRET. (a) GFP–N-Ras localizes to the plasma membrane and Golgi of Jurkat T cells. (b) Activation of exogenously expressed N-Ras only on the Golgi in Jurkat cells following cross-linking of the antigen receptor was observed by recruitment of GFP–RBD. (c) Bystander FRET. Jukat T cells were transiently cotransfected with KDELR–YFP, a molecule that is highly expressed on the Golgi apparatus, and RBD–CFP, serum starved and imaged alive before and after stimulation with antibodies to CD3 and CD28. The first panel shows the distribution of RBD–CFP that does not appear to change upon stimulation. The second panel shows the distribution of KDELR–YFP that likewise is constant. The third panel shows FRET images as sensitized emission (exitation at 458 nm, emission >560 nm). Note that a FRET signal is only apparent after stimulation and only on the Golgi. Arrowheads indicate the Golgi apparatus. Bars indicate 10 mM. Reprinted from [50] with permission from Elsevier. www.sciencedirect.com
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associated Ras in Raji B cells (A Mor and M Philips, unpublished). To document that endogenous Ras on the Golgi is activated downstream of PTKRs and TCR, Philips and colleagues employed a method called bystander FRET, whereby a FRET signal was detected on the Golgi apparatus between CFP–RBD and highly overexpressed CD8–YFP in cells expressing only endogenous Ras (Figure 1). Endogenous Ras activation on the Golgi was also observed by GFP–RBD recruitment upon overexpression of RasGRP1 [24]. Recently Rubio has developed an RBD-based probe that may be capable of revealing endogenous GTP–Ras by simple translocation of the probe [28]. This probe, designated GFP–RBD-3R59A,N64D, consists of three tandem RBD domains in which the affinity for GTP–Ras is somewhat diminished by mutation (wild-type tandem RBDs proved to be toxic). Using this probe the authors reported predominant activation on the PM. The discrepancy between the bystander FRET studies and those using GFP–RBD3-R59A,N64D remains to be resolved.
How are signals delivered to Ras on the Golgi? How does engagement of growth factor or antigen receptors at the cell surface transmit a signal to topologically remote Ras on the Golgi apparatus? Because activated Ras can enter the endosomal pathway on the cytosolic face of these organelles [29] and because retrograde traffic from endosomes to Golgi has been well-documented [30], the possibility of retrograde vesicular transport of activated Ras was explored. Under conditions in which vesicular traffic was inhibited, Ras activation on the Golgi was unimpeded [20]. This result suggested that Ras activation on the Golgi was under the control of diffusible mediators. The diffusible second messenger proved to be calcium: ras activation on the Golgi was sensitive to calcium chelation and to disruption of phospholipase Cg (PLCg). This evidence led to the discovery that RasGRP1, a GEF that is regulated by calcium and diacylgycerol (DAG) [31], is responsible for the activation of Ras in situ on the Golgi apparatus [27]. Interestingly, another calcium-responsive GEF, RasGRF, was shown to activate Ras only on the ER [25], suggesting that compartmentalized Ras signaling is driven by differential localization of GEFs. Overexpression of RasGRP1 led to Ras activation on the Golgi [24,32] and a dominant interfering mutant of RasGRP1 inhibited activation on this compartment [27]. Furthermore, silencing the RasGRP1 gene in Jurkat T cells with siRNA abolished recruitment of GFP–RBD to the Golgi [27]. Thus one pathway to Ras activation on the Golgi apparatus involves receptor/PLCg/Ca++ + DAG/RasGRP1 and thereby differs from the classical receptor/Grb2/SOS pathway that activates Ras on the PM (Figure 2). In addition, Shp2, via its effect on Src, regulates the pathway through phosphorylation of PLCg [33]. One important open question is how DAG levels are regulated on the Golgi to recruit Current Opinion in Cell Biology 2006, 18:162–167
RasGRP1 to that organelle. The Golgi is thought to be relatively rich in DAG and the content of this second messenger is regulated by phospholipase D activity and by sphingomyelin synthase [34], both of which are modulated by growth factor signaling. Chelating calcium or disrupting PLCg, Shp2 or Src not only blocked Ras activation on the Golgi but also inhibited the reversal of Ras activation at the PM [27]. These observations suggested that a calcium-sensitive GAP [31] operates at the PM to turn off Ras. Indeed, knockdown in Jurkat T cells with siRNA of the calcium-regulated Ras GAP CAPRI [35] resulted in Ras activation on both the PM and the Golgi following TCR stimulation [27], demonstrating that, in T cells, Ras activation on the PM is inhibited by CAPRI. Interestingly, since both CAPRI and RasGRP1 are activated by calcium, this single second messenger controls Ras signaling in opposite directions simultaneously on two different subcellular compartments (Figure 2). Recently, targeted disruption of CAPRI in mice has been shown to inhibit phagocytosis and innate immunity [36].
Retrograde transport of Ras to the Golgi Until recently, mature Ras proteins were thought to remain fixed at their target membranes. With a fixedRas model, the mechanisms discussed above can account for Ras activation in situ on the Golgi. However, recent paradigm-shifting studies have revealed that mature, fully processed Ras proteins are, in fact, in flux within the cell. The lability of the palmitate modification on N-Ras and an acylation/deacylation cycle has been known for almost two decades [37]. Recent photobleaching studies have demonstrated that depalmitoylated N-Ras and H-Ras are rapidly lost from the PM and transported in a retrograde fashion to the Golgi [26,38]. Using photoactivatable GFP, Rocks et al. demonstrated that the source of Golgiassociated Ras in a cell blocked for new protein synthesis is the PM [26]. Importantly, depalmitoylated Ras traffics to the Golgi by diffusing through the cytosol rather than by retrograde vesicular transport. The discovery of retrograde transport of Ras from the PM to the Golgi raises the possibility that GTP-bound Ras on the Golgi derives from activated Ras on the PM that remains GTPbound during retrograde transport. Support for this model was provided by Rocks et al., who demonstrated that disrupting the acylation/deacylation cycle by blocking palmitoylation on the Golgi with 2-bromopalmitate inhibited H-Ras activation on the Golgi [26]. This model predicts a pool of GTP-bound Ras in the cytosol, a requirement that has not been verified experimentally. It should be noted that Ras activation in situ on the Golgi via the RasGRP1 pathway and accumulation on the Golgi of GTP-bound Ras that derives from the PM are not mutually exclusive. Indeed, the absence of GTP-bound Ras on the Golgi in cells deficient in RasGRP1 [27] argues strongly for in situ activation. www.sciencedirect.com
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Figure 2
PLCg and RasGRP1 mediate Ras activation on the Golgi of T lymphocytes and CAPRI limits activation at the plasma membrane (PM). Activation of the TCR results in tyrosine phosphorylation of the z chain of the receptor by Src family kinases and the resulting phosphotyrosines serve to recruit ZAP-70 that in turn phosphorylates the scaffold protein LAT at multiple sites. Among the signaling molecules recruited to phosphorylated LAT is PLCg, which acts on phosphatidylinositol-4,5-bisphosphate in the PM to produce diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). Calcium liberated from internal stores by IP3 acts on the calcium- and DAG-sensitive Ras exchange factor RasGRP1 and causes it to translocate to the Golgi where DAG levels are relatively high. RasGRP1 activates Golgi-associated Ras on this compartment. Meanwhile, calcium also activates the Ras GAP CAPRI that translocates to the PM and downregulates any Ras that is activated on this compartment by the exchange factor SOS. Reprinted, with permission, from the Annual Review of Immunology, Volume 24 #2006 by Annual Reviews www.annualreviews.org.
Very recent work has revealed that the nonpalmitoylated isoform of Ras, K-Ras, can also traffic in a retrograde fashion from the PM to the Golgi apparatus [39,40]. Stimulation of rat hippocampal neurons with glutamate caused GFP–K-Ras to dissociate from the PM and associate with intracellular membranes including the Golgi apparatus and early endosomes. Evidence was presented for a calcium/calmodulin-dependent mechanism of retrograde transport of K-Ras but no consequences in terms of neuronal signaling were demonstrated [39]. An alternative mechanism for retrograde translocation of K-Ras was very recently reported. Serine 181 within the hypervariable domain of K-Ras serves as a site for PKC-mediated phosphorylation that partially neutralizes the polybasic region; this results in the loss of K-Ras from the PM via a mechanism termed the farnesyl-electrostatic switch because of its similarity to the myristoyl-electrostatic switch that regulates PM association of the MARCKS protein [41]. Interestingly, besides the Golgi and ER, the outer membrane of the mitochondria also serves as an acceptor site for phosphorylated K-Ras discharged from the PM. Surprisingly, phosphorylated K-Ras induces apoptosis in a Bcl-XL-dependent fashion [40]. Whether K-Ras on the Golgi takes part in the pro-apoptotic pathway remains to be determined. www.sciencedirect.com
Role of Ras signalling on the Golgi What is the physiologic significance of Ras signaling from intracellular membranes, in particular from the Golgi apparatus? Compartmentalized signaling, in theory, can increase the complexity of signaling by adding kinetically distinct outputs from a single pathway or by allowing activation of distinct downstream pathways. Indeed, compartmentalized signaling may help explain how a single regulatory molecule like Ras can control such a wide variety of cellular responses. Although the three Ras isoforms were originally thought to be functionally interchangeable, significant evidence has accumulated to contradict this view, not the least of which is the dramatic observation that whereas K-Ras is required for embryonic development [42], both H-Ras and N-Ras are dispensable [43]. At the level of cell signaling, it has been reported that K-Ras and H-Ras have differential efficiencies in activating their various effectors [44,45]. Since the Ras isoforms differ essentially only in their membrane targeting sequences, it seems reasonable to conclude that the differences in signaling are due to differences in subcellular localization. It should be noted that the different locations need not be on different organelles, as there is now abundant evidence for differential signaling of Ras from different microdomains within a single orgenelle, the PM [46]. Current Opinion in Cell Biology 2006, 18:162–167
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Direct evidence in support of the compartmentalized signaling model as it pertains to organelles has come from experiments in which transmembrane tethers were used to artificially and stringently target Ras proteins to various membrane compartments [20,24,25,47]. When oncogenic Ras was targeted to the ER (via the first transmembrane segment of the avian infectious bronchitis virus M1 protein) or to the Golgi (via the KDEL receptor), it retained full transforming activity, indicating that all the signaling events required for the complex cellular phenotype of transformation can be initiated from internal membranes [20,24]. This might suggest that Ras signaling from internal membranes is no different than that from the PM. However, quantitative differences in signal output could be detected. Whereas Golgi-associated oncogenic Ras activated the Erk and Akt pathways with potency equal to that of natively targeted Ras, the Jnk pathway was only weakly activated. Conversely, ERtethered oncogenic Ras was a potent activator of Jnk, but a relatively poor activator of the Erk and Akt pathways [20]. Recently these studies were repeated using a more stringent Golgi tether (a mutant of the KDEL receptor that has diminished capacity to cycle back to the ER) and a different result was obtained. Rather than Erk or Akt signaling, Golgi-associated Ras favored activation of the Ral pathway [47]. Compartmentalized Ras signaling appears to be conserved through evolution, since a recent study of Ras1 of Schizosaccharomyces pombe revealed that two pathways regulated by Ras1 are controlled from distinct membrane compartments (Onken, Philips and Chang, unpublished). Taken together, these studies demonstrate that the signaling output of Ras is determined to some extent by the intracellular platform from which signaling ensues. This view has been bolstered by the discovery of the differential localization of various MAPK scaffolds [48]. Indeed, Sef is a MEK/Erk scaffold that is restricted to the Golgi [49].
Conclusions Compartmentalized signaling is a new paradigm in the field of signal transduction. The hope is that by adding a spatial dimension to signaling we will learn how individual regulatory molecules can have outputs more complex than can be explained by their biochemistry alone. The recent discoveries about Ras activation on intracellular compartments, including the Golgi apparatus, described above have added important insight into this field. However, much remains to be learned. How is activation of Ras on the Golgi regulated? If RasGRP1 is primarily responsible, than what regulates the DAG content of Golgi membranes? If retrograde transport is critical, what regulates depalmitoylation and how is farnesylated Ras transported though the cytosol without a chaperone? Does Ras play a role in the classical vesicular transport function of the Golgi apparatus? Are Ras GAPs differentially localized in cells such that they too contribute to compartmentalized signaling? And, most Current Opinion in Cell Biology 2006, 18:162–167
important, what are the physiologic consequences for Ras signaling from the Golgi? Despite 24 years of seemingly exhaustive scrutiny, the Ras proteins still have more to teach us about fundamental concepts in cell biology.
Acknowledgement This work was supported by grants from the National Institutes of Health
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Current Opinion in Cell Biology 2006, 18:162–167
Current Opinion in Cell Biology 2006, 18:162–167 This review comes from a themed issue on Cell regulation Edited by Claude Prigent and Bruno Goud Available online 20th February 2006 0955-0674/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2006.02.004
Association of Ras proteins with the cytosolic leaflet of cellular membranes is required for biological activity [4– 6]. Ras proteins are synthesized as hydrophilic proteins on free polysomes in the cytosol and are targeted posttranslationally to cellular membranes by virtue of a series of modifications that include prenylation, proteolysis and carboxyl methylation [7]. These modifications are directed by a conserved C-terminal CAAX sequence where C is cysteine, A is usually an aliphatic amino acid and X is any amino acid [8]. In the case of Ras, the CAAX sequence is recognized by farnesyl transferase (FTase) [9], a cytosolic enzyme that catalyzes the addition of a 15carbon farnesyl isoprenoid to the CAAX cysteine via a stable thioether linkage [10]. Once prenylated, the Sisoprenyl CAAX moiety becomes a substrate for Rce1, an ER-restricted protease that cleaves the AAX sequence [7,11]. The newly C-terminal prenylcysteine is then recognized by a third enzyme, isoprenylcysteine carboxyl methyltransferase (Icmt), also restricted to the ER, that methylesterifies the carboxyl group [7,12]. The end result of these three modifications is to create a hydrophobic domain at the C-terminus that mediates membrane association. N-Ras, H-Ras and Kras4A, but not K-Ras4B, are further modified by the addition of one or two palmitic acids just upstream of the farnesylcysteine. A genetic screen in S. cerevisiae identified Erf2/Erf4 as the Ras palmitoyltransferase [13]. Sequence homology with Erf2/Erf4 revealed the human palmitoyltransferase to be a complex of the proteins DHHC9 and GCP16 [14], both of which localize to the Golgi apparatus [15].
Introduction Ras proteins are small GTPases that regulate cell growth, proliferation, differentiation and apoptosis. Ras genes were first recognized as the transforming principles of two retroviruses and mutated alleles of the cellular protooncogenes have been associated with a variety of human cancer [1]. Oncogenic Ras can induce the growth and transformation of cells both in vitro and in vivo [2]. Ras proteins are founding members of a large superfamily of monomeric GTPases that function as molecular switches, cycling between inactive, GDP-bound and active, GTPbound forms. Guanine nucleotide-exchange factors (GEFs) mediate signal-induced activation of Ras by stimulating the exchange of GDP for GTP; this induces a conformational change in the protein [3] that permits interaction with downstream effectors such as Raf-1, PI3K, and RalGDS. The active state of Ras is self-limited by its intrinsic GTPase activity and this relatively weak activity is greatly accelerated by GTPase activating proteins (GAPs). Mammalian genomes encode three ras genes that give rise to four ubiquitously expressed gene products: N-Ras, H-Ras, K-Ras4A and K-Ras4B. Current Opinion in Cell Biology 2006, 18:162–167
The post-translational modifications to the C-terminal CAAX motif of Ras were originally thought to target nascent cytosolic Ras proteins directly to the plasma membrane (PM). The discovery that the enzymes that further modify prenylated proteins are restricted to the ER or Golgi [12,14–17] suggested that nascent Ras must traffic to the PM via the endomembrane. This prediction was verified by studying the trafficking of green fluorescent protein (GFP)-tagged Ras [5,18]. Once fully modified on the endomembrane, the subsequent transfer of Ras proteins to the PM requires a second signal found in the C-terminal hypervariable regions of Ras proteins immediately adjacent to their CAAX motifs. For N-Ras and H-Ras this signal consists of one or two cysteine residues that serve as sites of palmitoylation. For KRas4B, the signal consists of a polybasic region rich in lysine residues [6,19]. Whereas prenylation alone serves as a relatively weak membrane anchor, palmitoylation markedly increases affinity for membranes, trapping NRas and H-Ras in the phospholipid bilayer. The positively charged polybasic region of K-Ras is thought to www.sciencedirect.com
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associate with the negatively charged head groups of the inner leaflet phospholipids of the PM. Because Ras GTPases link receptors, such as protein tyrosine kinase receptors (PTKRs), that traverse the PM with cytoplasmic effector pathways that regulate cellular function, it is intuitive to think that Ras signaling occurs at the PM. The discovery that Ras proteins visit the endomembrane en route to the PM, and that at steady-state the palmitoylated isoforms of Ras are highly expressed on the Golgi apparatus, led to the hypothesis that Ras signaling may also occur on intracellular compartments. This hypothesis has been tested in living cells with fluorescent probes and shown to be correct: Ras and/ or MAPK signaling originates from the cytoplasmic face of endosomes, the ER, the Golgi apparatus and mitochondria as well as from the PM. In this review, we will cover the most recent advances in understanding Ras signaling from the Golgi apparatus.
Ras activity on the Golgi The observation that activated Ras accumulates on the Golgi was made possible by the advent of genetically encoded fluorescent probes that are capable of reporting where and when Ras becomes loaded with GTP in living cells [20–22]. The first of these was the founding member of the so-called Raichu probes developed by Matsuda and colleagues. Raichu-Ras is a FRET-based probe where Ras and the Ras binding domain (RBD) of Raf-1 are in cis on a single polypeptide that is flanked by CFP and YFP (cyan and yellow fluorescent proteins) such that, when the exogenous Ras is GTP-bound, the molecule assumes a hairpin configuration and a FRET signal is generated [21]. Raichu-Ras is targeted to the PM with the 20-aminoacid C-terminus of K-Ras such that it is spatially biased in
its reporting. Moreover, because the probe incorporates both the GTPase and the cognate RBD, which are therefore overexpressed, rather than reporting local Ras activation it actually reports the local balance of GEFs and GAPs [23]. As expected by its PM targeting, Raichu-Ras did not report Ras activation on the Golgi [21]. Perhaps the most informative probe for GTP-bound Ras is the simplest, consisting of the RBD fused to GFP. This probe reports Ras activation in a spatiotemporal manner by translocating from the cytosol and nucleoplasm to membrane compartments upon which Ras becomes activated (Figure 1). Among the advantages of this probe is that it is not itself targeted in any way, meaning that it is spatially unbiased. The major disadvantage of this probe is that unless a GEF is overexpressed [24], it is not sensitive enough to detect endogenous GTP-bound Ras and therefore requires overexpression of wild-type Ras proteins. Using GFP–RBD, Chiu et al. showed that upon stimulation of fibroblasts with epidermal growth factor (EGF), Ras became activated at both the PM and the Golgi [20]. These observations have now been confirmed by several laboratories [24,25,26]. Interestingly, the kinetics of activation differed between the two compartments: whereas recruitment of GFP–RBD to the PM was rapid and transient (1–3 min onset, 10 min duration), activation of Ras on the Golgi was delayed and sustained (onset 10– 20 min, duration >60 min). The kinetics and relative extent of compartment-specific activation is cell-typespecific. In Jurkat T cells stimulated through the antigen receptor (TCR), activation on the Golgi was rapid (1– 3 min) and, surprisingly, no activation on the PM was observed [27]. Ras activation exclusively on the Golgi downstream of TCR signaling was also observed in primary T cells, as was constitutive activation of Golgi-
Figure 1
Detection of endogenous Ras activation on the Golgi of Jurkat T cells using bystander FRET. (a) GFP–N-Ras localizes to the plasma membrane and Golgi of Jurkat T cells. (b) Activation of exogenously expressed N-Ras only on the Golgi in Jurkat cells following cross-linking of the antigen receptor was observed by recruitment of GFP–RBD. (c) Bystander FRET. Jukat T cells were transiently cotransfected with KDELR–YFP, a molecule that is highly expressed on the Golgi apparatus, and RBD–CFP, serum starved and imaged alive before and after stimulation with antibodies to CD3 and CD28. The first panel shows the distribution of RBD–CFP that does not appear to change upon stimulation. The second panel shows the distribution of KDELR–YFP that likewise is constant. The third panel shows FRET images as sensitized emission (exitation at 458 nm, emission >560 nm). Note that a FRET signal is only apparent after stimulation and only on the Golgi. Arrowheads indicate the Golgi apparatus. Bars indicate 10 mM. Reprinted from [50] with permission from Elsevier. www.sciencedirect.com
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164 Cell regulation
associated Ras in Raji B cells (A Mor and M Philips, unpublished). To document that endogenous Ras on the Golgi is activated downstream of PTKRs and TCR, Philips and colleagues employed a method called bystander FRET, whereby a FRET signal was detected on the Golgi apparatus between CFP–RBD and highly overexpressed CD8–YFP in cells expressing only endogenous Ras (Figure 1). Endogenous Ras activation on the Golgi was also observed by GFP–RBD recruitment upon overexpression of RasGRP1 [24]. Recently Rubio has developed an RBD-based probe that may be capable of revealing endogenous GTP–Ras by simple translocation of the probe [28]. This probe, designated GFP–RBD-3R59A,N64D, consists of three tandem RBD domains in which the affinity for GTP–Ras is somewhat diminished by mutation (wild-type tandem RBDs proved to be toxic). Using this probe the authors reported predominant activation on the PM. The discrepancy between the bystander FRET studies and those using GFP–RBD3-R59A,N64D remains to be resolved.
How are signals delivered to Ras on the Golgi? How does engagement of growth factor or antigen receptors at the cell surface transmit a signal to topologically remote Ras on the Golgi apparatus? Because activated Ras can enter the endosomal pathway on the cytosolic face of these organelles [29] and because retrograde traffic from endosomes to Golgi has been well-documented [30], the possibility of retrograde vesicular transport of activated Ras was explored. Under conditions in which vesicular traffic was inhibited, Ras activation on the Golgi was unimpeded [20]. This result suggested that Ras activation on the Golgi was under the control of diffusible mediators. The diffusible second messenger proved to be calcium: ras activation on the Golgi was sensitive to calcium chelation and to disruption of phospholipase Cg (PLCg). This evidence led to the discovery that RasGRP1, a GEF that is regulated by calcium and diacylgycerol (DAG) [31], is responsible for the activation of Ras in situ on the Golgi apparatus [27]. Interestingly, another calcium-responsive GEF, RasGRF, was shown to activate Ras only on the ER [25], suggesting that compartmentalized Ras signaling is driven by differential localization of GEFs. Overexpression of RasGRP1 led to Ras activation on the Golgi [24,32] and a dominant interfering mutant of RasGRP1 inhibited activation on this compartment [27]. Furthermore, silencing the RasGRP1 gene in Jurkat T cells with siRNA abolished recruitment of GFP–RBD to the Golgi [27]. Thus one pathway to Ras activation on the Golgi apparatus involves receptor/PLCg/Ca++ + DAG/RasGRP1 and thereby differs from the classical receptor/Grb2/SOS pathway that activates Ras on the PM (Figure 2). In addition, Shp2, via its effect on Src, regulates the pathway through phosphorylation of PLCg [33]. One important open question is how DAG levels are regulated on the Golgi to recruit Current Opinion in Cell Biology 2006, 18:162–167
RasGRP1 to that organelle. The Golgi is thought to be relatively rich in DAG and the content of this second messenger is regulated by phospholipase D activity and by sphingomyelin synthase [34], both of which are modulated by growth factor signaling. Chelating calcium or disrupting PLCg, Shp2 or Src not only blocked Ras activation on the Golgi but also inhibited the reversal of Ras activation at the PM [27]. These observations suggested that a calcium-sensitive GAP [31] operates at the PM to turn off Ras. Indeed, knockdown in Jurkat T cells with siRNA of the calcium-regulated Ras GAP CAPRI [35] resulted in Ras activation on both the PM and the Golgi following TCR stimulation [27], demonstrating that, in T cells, Ras activation on the PM is inhibited by CAPRI. Interestingly, since both CAPRI and RasGRP1 are activated by calcium, this single second messenger controls Ras signaling in opposite directions simultaneously on two different subcellular compartments (Figure 2). Recently, targeted disruption of CAPRI in mice has been shown to inhibit phagocytosis and innate immunity [36].
Retrograde transport of Ras to the Golgi Until recently, mature Ras proteins were thought to remain fixed at their target membranes. With a fixedRas model, the mechanisms discussed above can account for Ras activation in situ on the Golgi. However, recent paradigm-shifting studies have revealed that mature, fully processed Ras proteins are, in fact, in flux within the cell. The lability of the palmitate modification on N-Ras and an acylation/deacylation cycle has been known for almost two decades [37]. Recent photobleaching studies have demonstrated that depalmitoylated N-Ras and H-Ras are rapidly lost from the PM and transported in a retrograde fashion to the Golgi [26,38]. Using photoactivatable GFP, Rocks et al. demonstrated that the source of Golgiassociated Ras in a cell blocked for new protein synthesis is the PM [26]. Importantly, depalmitoylated Ras traffics to the Golgi by diffusing through the cytosol rather than by retrograde vesicular transport. The discovery of retrograde transport of Ras from the PM to the Golgi raises the possibility that GTP-bound Ras on the Golgi derives from activated Ras on the PM that remains GTPbound during retrograde transport. Support for this model was provided by Rocks et al., who demonstrated that disrupting the acylation/deacylation cycle by blocking palmitoylation on the Golgi with 2-bromopalmitate inhibited H-Ras activation on the Golgi [26]. This model predicts a pool of GTP-bound Ras in the cytosol, a requirement that has not been verified experimentally. It should be noted that Ras activation in situ on the Golgi via the RasGRP1 pathway and accumulation on the Golgi of GTP-bound Ras that derives from the PM are not mutually exclusive. Indeed, the absence of GTP-bound Ras on the Golgi in cells deficient in RasGRP1 [27] argues strongly for in situ activation. www.sciencedirect.com
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Figure 2
PLCg and RasGRP1 mediate Ras activation on the Golgi of T lymphocytes and CAPRI limits activation at the plasma membrane (PM). Activation of the TCR results in tyrosine phosphorylation of the z chain of the receptor by Src family kinases and the resulting phosphotyrosines serve to recruit ZAP-70 that in turn phosphorylates the scaffold protein LAT at multiple sites. Among the signaling molecules recruited to phosphorylated LAT is PLCg, which acts on phosphatidylinositol-4,5-bisphosphate in the PM to produce diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). Calcium liberated from internal stores by IP3 acts on the calcium- and DAG-sensitive Ras exchange factor RasGRP1 and causes it to translocate to the Golgi where DAG levels are relatively high. RasGRP1 activates Golgi-associated Ras on this compartment. Meanwhile, calcium also activates the Ras GAP CAPRI that translocates to the PM and downregulates any Ras that is activated on this compartment by the exchange factor SOS. Reprinted, with permission, from the Annual Review of Immunology, Volume 24 #2006 by Annual Reviews www.annualreviews.org.
Very recent work has revealed that the nonpalmitoylated isoform of Ras, K-Ras, can also traffic in a retrograde fashion from the PM to the Golgi apparatus [39,40]. Stimulation of rat hippocampal neurons with glutamate caused GFP–K-Ras to dissociate from the PM and associate with intracellular membranes including the Golgi apparatus and early endosomes. Evidence was presented for a calcium/calmodulin-dependent mechanism of retrograde transport of K-Ras but no consequences in terms of neuronal signaling were demonstrated [39]. An alternative mechanism for retrograde translocation of K-Ras was very recently reported. Serine 181 within the hypervariable domain of K-Ras serves as a site for PKC-mediated phosphorylation that partially neutralizes the polybasic region; this results in the loss of K-Ras from the PM via a mechanism termed the farnesyl-electrostatic switch because of its similarity to the myristoyl-electrostatic switch that regulates PM association of the MARCKS protein [41]. Interestingly, besides the Golgi and ER, the outer membrane of the mitochondria also serves as an acceptor site for phosphorylated K-Ras discharged from the PM. Surprisingly, phosphorylated K-Ras induces apoptosis in a Bcl-XL-dependent fashion [40]. Whether K-Ras on the Golgi takes part in the pro-apoptotic pathway remains to be determined. www.sciencedirect.com
Role of Ras signalling on the Golgi What is the physiologic significance of Ras signaling from intracellular membranes, in particular from the Golgi apparatus? Compartmentalized signaling, in theory, can increase the complexity of signaling by adding kinetically distinct outputs from a single pathway or by allowing activation of distinct downstream pathways. Indeed, compartmentalized signaling may help explain how a single regulatory molecule like Ras can control such a wide variety of cellular responses. Although the three Ras isoforms were originally thought to be functionally interchangeable, significant evidence has accumulated to contradict this view, not the least of which is the dramatic observation that whereas K-Ras is required for embryonic development [42], both H-Ras and N-Ras are dispensable [43]. At the level of cell signaling, it has been reported that K-Ras and H-Ras have differential efficiencies in activating their various effectors [44,45]. Since the Ras isoforms differ essentially only in their membrane targeting sequences, it seems reasonable to conclude that the differences in signaling are due to differences in subcellular localization. It should be noted that the different locations need not be on different organelles, as there is now abundant evidence for differential signaling of Ras from different microdomains within a single orgenelle, the PM [46]. Current Opinion in Cell Biology 2006, 18:162–167
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Direct evidence in support of the compartmentalized signaling model as it pertains to organelles has come from experiments in which transmembrane tethers were used to artificially and stringently target Ras proteins to various membrane compartments [20,24,25,47]. When oncogenic Ras was targeted to the ER (via the first transmembrane segment of the avian infectious bronchitis virus M1 protein) or to the Golgi (via the KDEL receptor), it retained full transforming activity, indicating that all the signaling events required for the complex cellular phenotype of transformation can be initiated from internal membranes [20,24]. This might suggest that Ras signaling from internal membranes is no different than that from the PM. However, quantitative differences in signal output could be detected. Whereas Golgi-associated oncogenic Ras activated the Erk and Akt pathways with potency equal to that of natively targeted Ras, the Jnk pathway was only weakly activated. Conversely, ERtethered oncogenic Ras was a potent activator of Jnk, but a relatively poor activator of the Erk and Akt pathways [20]. Recently these studies were repeated using a more stringent Golgi tether (a mutant of the KDEL receptor that has diminished capacity to cycle back to the ER) and a different result was obtained. Rather than Erk or Akt signaling, Golgi-associated Ras favored activation of the Ral pathway [47]. Compartmentalized Ras signaling appears to be conserved through evolution, since a recent study of Ras1 of Schizosaccharomyces pombe revealed that two pathways regulated by Ras1 are controlled from distinct membrane compartments (Onken, Philips and Chang, unpublished). Taken together, these studies demonstrate that the signaling output of Ras is determined to some extent by the intracellular platform from which signaling ensues. This view has been bolstered by the discovery of the differential localization of various MAPK scaffolds [48]. Indeed, Sef is a MEK/Erk scaffold that is restricted to the Golgi [49].
Conclusions Compartmentalized signaling is a new paradigm in the field of signal transduction. The hope is that by adding a spatial dimension to signaling we will learn how individual regulatory molecules can have outputs more complex than can be explained by their biochemistry alone. The recent discoveries about Ras activation on intracellular compartments, including the Golgi apparatus, described above have added important insight into this field. However, much remains to be learned. How is activation of Ras on the Golgi regulated? If RasGRP1 is primarily responsible, than what regulates the DAG content of Golgi membranes? If retrograde transport is critical, what regulates depalmitoylation and how is farnesylated Ras transported though the cytosol without a chaperone? Does Ras play a role in the classical vesicular transport function of the Golgi apparatus? Are Ras GAPs differentially localized in cells such that they too contribute to compartmentalized signaling? And, most Current Opinion in Cell Biology 2006, 18:162–167
important, what are the physiologic consequences for Ras signaling from the Golgi? Despite 24 years of seemingly exhaustive scrutiny, the Ras proteins still have more to teach us about fundamental concepts in cell biology.
Acknowledgement This work was supported by grants from the National Institutes of Health
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Current Opinion in Cell Biology 2006, 18:162–167