Correspondence to Marc Fivaz: mfivaz{at}stanford.edu; or Tobias Meyer: Tobias1{at}stanford.edu
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Introduction |
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In addition to an essential role in regulating cell growth and differentiation, Ras signaling has more recently been linked to a wide range of neuronal functions including synaptic and behavioral plasticity (for review see Thomas and Huganir, 2004). Pharmacological and genetic manipulations of the Ras/MAPK cascade provided evidence for a role in learning and memory (Di Cristo et al., 2001; Wu et al., 2001; Kelleher et al., 2004), long-term potentiation (Di Cristo et al., 2001; Kelleher et al., 2004), and other forms of synaptic plasticity (Huang et al., 2000; Wu et al., 2001). Neuronal activation of Ras by membrane depolarization or glutamatergic signaling does not operate "classically" through tyrosine kinases and adaptors, but shows a strong dependence on Ca2+ influx through the N-methyl-D-aspartate receptor (NMDA-R) or voltage-gated Ca2+ channels (Cullen and Lockyer, 2002). This Ca2+-dependent component in Ras/MAPK signaling is thought to be important in relaying excitatory synaptic inputs to gene transcription in the nucleus (Dolmetsch et al., 2001), a process required for long-term synaptic remodeling and neuronal survival (Dolmetsch, 2003).
Although considerable evidence points to an essential role of Ras signaling in a variety of neuronal functions, little is known about isoform-specific functions and subcellular localization in neurons. Using a systematic approach to characterize the role of different membrane-anchoring motifs, we here report that the prenyl-polybasic targeting motifs of KRas and Rap1a dictate rapid, reversible and Ca2+-dependent subcellular redistribution in response to glutamatergic signaling. Activity-dependent translocation of KRas to endomembranes is isoform specific and occurs via a shuttling mechanism through the cytoplasm controlled by Ca2+/CaM. We provide evidence that a fraction of KRas relocalized to endomembranes remains in a signaling competent form, suggesting that this activity-dependent translocation process may spatially segregate and modulate KRas and HRas signaling activities in neurons.
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Results |
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Neuronal activity triggers reversible translocation of CFP-Kras-tail and CFP-Rap1a-tail to intracellular membranes
We then tested whether the distribution of these probes was sensitive to neuronal activity. Stimulation of hippocampal neurons by bath application of 50 µM glutamate had no effect on their distribution with the exception of CFP-KRas-tail and CFP-Rap1a-tail. Glutamate stimulation led to a striking redistribution of CFP-KRas-tail from the PM to a perinuclear (PN) membrane compartment and dendritic vesicles (Fig. 1 a; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200409157/DC1). Release of CFP-KRas-tail from the PM occurred throughout the somatodendritic region with fast kinetics (t1/2 2.5 min) and preceded the accumulation of the probe in intracellular membranes (t1/2
4.5 min) (Fig. 1, a and c). Interestingly, the CFP-HRas-tail probe (Fig. 1, d and e) did not translocate in response to glutamate. CFP-Rap1a-tail, which exhibits a membrane-interacting motif structurally related to KRas-tail (Table S1), also redistributed intracellularly upon glutamate application. Redistribution was, however, less pronounced (Fig. 1 g) and occurred with slower kinetics (Fig. 1 f). Neuronal activation elicited by NMDA, high K+ membrane depolarization, or selective stimulation of synaptic NMDA-Rs (Lu et al., 2001) also induced intracellular redistribution of CFP-KRas-tail and CFP-Rap1a-tail, albeit with slightly different kinetics (unpublished data). Importantly, transient application of glutamate (typically a 23-min pulse) led to a fully reversible translocation of CFP-KRas-tail (Fig. 2; Video 2) to intracellular vesicles (in the soma and the dendritic arbor) and back to the PM, as shown by a ratiometric analysis using a reference PM marker (see Materials and methods).
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Cytoplasmic shuttling of the KRas-tail probe to Golgi and early endosomal membranes
Dual color measurements using a Golgi marker (CFP-GalTase) showed that the KRas-tail probe partially colocalizes with the Golgi complex after translocation (Fig. 3 a). Pretreatment with brefeldin A (a fungal metabolite that disrupts the Golgi) only partially inhibited KRas-tail accumulation in PN membranes (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200409157/DC1), suggesting that CFP-KRas-tail also localizes to another juxtanuclear compartment. Consistently, CFP-Kras-tail also partially overlapped with recycling endosomes loaded with Alexa 546labeled transferrin (alexa546-Tf) (Fig. 4 a). In dendritic processes, KRas-tail clearly redistributed to early endosomes, as shown by colocalization with the early endosome marker (FYVE)2-YFP (Fig. 3 b; Video 3) (Gillooly et al., 2000).
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Full-length KRas exhibits the same translocation response
We then examined whether the full-length CFP-conjugated KRas protein translocates to intracellular membranes in response to glutamate. Fig. 5 (a and b) shows that CFP-KRas undergoes glutamate-induced translocation to PN membranes with kinetics indistinguishable to that of the KRas-tail probe (t1/2 4.5 min). CFP-tagged constitutively active (Q61L) and dominant-negative (S17N) mutants of KRas redistributed intracellularly with similar kinetics (Fig. 5, a and b), indicating that the translocation process is independent of the nucleotide state of the small GTPase. As observed for the KRas-tail and HRas-tail probes, translocation of the full-length protein occurred for KRas, but not for HRas (Fig. 5, a and c). Together, these results show that the membrane targeting motif of KRas is necessary and sufficient for glutamate-induced translocation of the full-length GTPase, and that translocation is selective for KRas over HRas. An interesting corollary of these data is that neuronal activity leads to a transient spatial segregation of the KRas and HRas isoforms.
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Intracellular redistribution of KRas activity in response to glutamatergic signaling
Finally, to investigate the role of KRas translocation in neuronal Ras signaling, we made use of a fluorescent reporter for active, GTP-bound Ras. The probe consists of the Ras-binding domain (RBD) of Raf-1 fused to CFP (RBD-CFP) (Chiu et al., 2002). When cotransfected with YFP-KRas, RBD-CFP localized diffusively in the cytoplasm in resting cells (Fig. 10 a, first panel). Addition of 50 µM NMDA triggered recruitment of RBD-CFP to the PM (Fig. 10 a) within tens of seconds, indicating rapid activation of Ras in response to neuronal activity. Recruitment of RBD-CFP to the PM did not occur in cells expressing RBD-CFP only (unpublished data), consistent with the previous observation that the RBD probe is not sensitive enough to report endogenous activation of Ras (Chiu et al., 2002). As YFP-KRas translocated to PN membranes, RBD-CFP redistributed and colocalized with intracellular structures positive for YFP-KRas (Fig. 10 a), indicating that a fraction of translocated KRas remained in a GTP-bound, signaling-competent form after it translocated to internal membranes. The recruitment of RBD to intracellular compartments was delayed compared with recruitment to the PM and persisted even after most of YFP-KRas (and RBD-CFP) had left the PM (Fig. 10 b), suggesting that KRas translocation leads to sustained internal KRas activity in response to neuronal stimulation.
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Discussion |
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Activity-dependent reversible membrane interactions mediated by the polybasic-prenyl membrane targeting motif
Prenyl-based PM targeting motifs of Ras-like small GTPases are generally viewed as stable, nonregulated PM anchors, unlike other lipid modifications such as S-palmitoylation or N-myristoylation, which can reversibly interact with membranes (for reviews see Ames et al., 1997; Bijlmakers and Marsh, 2003; Fivaz and Meyer, 2003). Our work now shows that prenylation, in the context of an adjacent polybasic region, can reversibly transfer proteins from the PM to intracellular membranes within minutes, indicating that prenyl-based membrane interactions are more dynamic than previously envisioned. Reversible membrane interactions of KRas in neurons is in agreement with studies showing that KRas can dissociate from membranes in vitro (Leventis and Silvius, 1998; Roy et al., 2000).
Reversible membrane interactions via a Ca2+/CaM-regulated lipid-dependent switch
Our work indicates that reversible membrane interactions mediated by the PM targeting motifs of KRas and Rap1a is regulated by Ca2+/CaM. First, CaM binds to the COOH-terminal membrane anchors of KRas and Rap1a (but not Hras) and pulls these probes off the membrane in vitro (Fig. 9, a and b). Second, glutamate stimulation leads to recruitment of Ca2+/CaM to the COOH-terminal tail of KRas at the PM, before KRas translocation (Fig. 8). Finally, KRas-tail translocation in vivo is inhibited by the CaM antagonist w-7 (Fig. 7). Together, these results support the notion that Ca2+/CaM sequesters the polybasic-prenyl motif of KRas and Rap1a and extracts these small GTPases from the PM. The observed difference in kinetics of translocation between CFP-KRas-tail and CFP-Rap1a-tail may reflect the difference in length between the farnesyl (C15) and generanylgeranyl (C20) lipid moieties (the longer lipid anchor presumably confers more affinity for the PM). Destabilization of this membrane interaction by Ca2+/CaM could result from disruption of electrostatic interactions between the polybasic region and negatively charged phospholipids of the PM and/or direct hydrophobic interactions of Ca2+/CaM with the prenyl group (see below). A role of CaM as a modulator of reversible proteinmembrane interactions has been previously described for the myristoylated alanine-rich C kinase substrate (MARCKS) protein. The polybasic effector domain of MARCKS interacts electrostatically with acidic lipids (Rusu et al., 2004) and is a good substrate for Ca2+/CaM. Binding of Ca2+/CaM to the effector domain pulls the peptide off membranes (Arbuzova et al., 1997). By analogy with this switch mechanism initially put forward by McLaughlin and Aderem (1995), we would like to propose that the polybasic-prenyl targeting motif of KRas and Rap1a acts as a molecular switch that controls reversible membrane interactions of KRas and Rap1a through Ca2+/CaM.
What is the Ca2+/CaM binding site in the COOH-terminal tails of KRas and Rap1a? The polybasic region is reminiscent of a class of Ca2+/CaM-binding motifs that consists of a high density of basic residues (50% of the sequence) alternating with hydrophobic residues (MARCKS is an example of this basic Ca2+/CaM-binding motif [Aderem, 1992; Yamauchi et al., 2003]). However, there is no hydrophobic residue in the COOH-terminal tail of KRas, and only one valine residue in the COOH-terminal tail of Rap1a (see Table S1). Because hydrophobic interactions are central to Ca2+/CaM target recognition (Yamniuk and Vogel, 2004), we believe it is unlikely that the polybasic domain of KRas and Rap1a acts as a Ca2+/CaM-binding motif on its own. Within the last few years, several N-myristoylated proteins have been shown to interact with Ca2+/CaM, defining a novel potential CaM-binding motif (Takasaki et al., 1999; Hayashi et al., 2002, 2004; Matsubara et al., 2003, 2004). A recent crystal structure of CAP23/NAP bound to CaM revealed that the myristoyl moiety penetrates in the Ca2+-exposed hydrophobic groove of CaM (Matsubara et al., 2004), in a way reminiscent of the hydrophobic pocket of Rho-GDI for isoprenylated cdc42 (Gosser et al., 1997). Consistent with a direct role of the prenyl moiety in CaM recognition, we found that substitution of the prenylated cysteine to an alanine in the CaaX motif significantly reduced binding of CFP-KRas-tail to Ca2+/CaM (Fig. 9 a).
The inability of HRas to interact with Ca2+/CaM could be due to the absence of a polybasic region and (or) the presence of additional palmitoyl modifications that are unlikely to fit in the Ca2+/CaM hydrophobic pocket. Because Ca2+/CaM binding to the COOH-terminal tail of KRas and Rap1a does not seem to rely on a particular amino acid sequence, but rather on the presence of a polybasic-prenyl combination, Ca2+/CaM-mediated reversible membrane interactions may potentially apply to a sizable subgroup of small GTPases and other proteins. In support of this prediction, CaM has been shown to interact in vitro with a number of small GTPases that exhibit a polybasic-prenyl motif, such as Rab3A (Park et al., 1997), Rab3B (Sidhu and Bhullar, 2001), RalA (Wang et al., 1997), RalB (Clough et al., 2002), and KRas (Sidhu et al., 2003. In addition, CaM has also been reported to dissociate Rab3A (Park et al., 1997; Villalonga et al., 2001) and KRas (Sidhu et al., 2003) from membranes in vitro.
Ca2+/CaM-dependent translocation of KRas: a process related to GDI function
Ca2+/CaM-mediated retrieval of KRas and Rap1a from the PM bears some similarities with GDP dissociation inhibitor (GDI)mediated recycling of Rab and Rho family members (Wu et al., 1996; Olofsson, 1999). Ca2+/CaM, like GDI, extracts small GTPases from membranes and presumably shields the prenyl moiety from the hydrophilic environment of the cytoplasm. Ca2+/CaM-mediated reversible translocation of KRas to the Golgi is therefore conceptually analogous to recycling of Rab GTPases from a target to a donor membrane compartment during one cycle of vesicular traffic. However, whereas GDI operates on the inactive GDP-bound form of the GTPase only, CaM function is triggered by Ca2+, and is therefore independent of the nucleotide state of the small GTPase (Fig. 5). Whether CaM also plays a role in unloading KRas onto intracellular membranes is presently unknown.
Recycling of CFP-KRas-tail back to the PM
We have shown that upon transient glutamate stimulation, CFP-KRas-tail translocation to intracellular membranes is fully reversible (Fig. 2), and that recycling of CFP-KRas-tail back to the PM correlates with a return of Ca2+ to basal levels (Fig. 6 c). One plausible interpretation of these data is that upon Ca2+ decrease, CaM releases from the KRas tail, allowing the probe to recycle back from the intracellular membranes to the PM. The mechanism of transport back to the PM may share some similarities with delivery of newly synthesized KRas to the PM.
Newly synthesized Ras isoforms (i.e., KRas, NRas, and HRas) are first delivered to ER/Golgi membranes where maturation of the prenyl modification proceeds, through endoproteolytic removal of AAX, and carboxymethylation of the COOH-terminal isoprenylcysteine (Choy et al., 1999; Fu and Casey, 1999). Targeting of matured Ras to the PM relies on accessory signals; palmitoylation for HRas/NRas and a polybasic region for KRas (Choy et al., 1999; Roy et al., 2000). Intriguingly, whereas HRas/NRas delivery to the PM occurs via vesicular transport through the secretory pathway, KRas targeting to the PM operates via a yet-undefined, presumably nonvesicular pathway (Thissen et al., 1997; Apolloni et al., 2000; Silvius, 2002), possibly by simple diffusion down an electrostatic gradient (greater negative charge density at the PM, relative to intracellular membranes), where PM targeting would be essentially driven by electrostatic interactions of the KRas polybasic domain with negatively charged phospholipids. Likewise, the polybasic region of KRas (now free of Ca2+/CaM) may be responsible for recycling the small GTPase from intracellular membranes back to the PM via a similar mechanism.
Activity-dependent translocation of KRas: implications for neuronal Ras signaling
Work by Chiu et al. (2002) and Bivona et al. (2003) has shown that in fibroblasts, T cells, and PC12 cells, ER/Golgi-localized HRas signaling is engaged in response to growth receptors or T cell receptor activation at the PM. In their study, activation of HRas in the Golgi was shown to be the result of a Ca2+- and PLC1- dependent recruitment of the Ras guanine nucleotide exchange factor RasGRP1 to Golgi membranes (Bivona et al., 2003). We have now identified an alternative mechanism by which Ras signaling can be carried to intracellular membranes in response to neuronal excitatory inputs. In this case, the presence of active, GTP-bound KRas in endomembranes relies on Ca2+/CaM-dependent translocation of KRas, rather than activation of prelocalized HRas by a diffusible messenger.
A model that integrates translocation of KRas and Ras/MAPK signaling at a glutamatergic synapse is shown in Fig. 10 c. Activity-dependent Ca2+ influx through the NMDA-R or L-type voltage-gated Ca2+ channels (unpublished data) leads to rapid (within seconds) Ras activation at the PM by means of Ca2+- or Ca2+/CaM-dependent Ras GEFs (Krapivinsky et al., 2003). KRas (but not HRas) then undergoes Ca2+/CaM-dependent translocation to endomembranes (within minutes) and further engages signaling processes from intracellular sites. One speculative role of KRas translocation, in the context of activity-dependent gene transcription, would be to facilitate the relay of local Ca2+ elevations in dendritic spines to the nucleus by localizing the MAPK pathway in closer proximity to the nucleus.
In conclusion, we have identified what is to our knowledge the first example of a signaling-induced translocation of a Ras family member. The mechanisms that underlie this translocation process are potentially applicable to a number of other signaling proteins, and may provide a molecular and cellular basis for signaling specificity of a variety of lipid-modified signaling molecules in neurons.
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Materials and methods |
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Cell culture transfection
Rat hippocampal neurons were prepared as previously described (Fink et al., 2003). Neurons were transfected at 7 d in vitro (DIV) using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions and imaged between DIV 10 and DIV 15. HeLa cells were cultured in DME (GIBCO BRL) in 10% FCS and transfected with FuGENE (Roche) according to the manufacturer's instructions.
Live-cell confocal microscopy and immunocytochemistry
Live-cell dual color measurements were performed on a spinning-disc confocal microscope. CFP and YFP excitations were obtained by the 442-nm line of a helium-cadmium laser (100 mM; Kimmon Electrics) and the 514-nm line of an argon laser (300 mW; Melles Griot), respectively. The beams of the two lasers were merged onto a beam combiner dichroic (Chroma Technology Corp.), homogenized with a holographic laser diffuser and focused into the confocal scan head (Yokogawa, McBain) equipped with a dual CFP/YFP dichroic and mounted on the side port of an inverted microscope (model IX-71; Olympus). Fluorescence was recorded at 480 nm (CFP) and 530 nm (YFP) with bandpass (40-nm half-bandwith) and longpass interference filters, respectively, mounted in a Lambda 10-2 filter wheel (Sutter Instrument Co.). Images were captured with a CCD camera (Cool SNAP HQ; Roper Scientific), driven by MetaMorph 6.1 (Universal Imaging Corp.). Neurons were imaged in extracellular buffer EB (25 mM Hepes, pH 7.40, 150 mM NaCl, 1.3 mM CaCl2, 1.3 mM MgCl2, 5 mM KCl, and 33 mM glucose) on a heated stage (33-35C), using a 40x (NA 1.35) objective. Neurons were processed for immunocytochemistry as described elsewhere (Fink et al., 2003). In brief, neurons were fixed in 4% PFA and 4% sucrose, and were permeabilized with 0.l% Triton X-100 for 5 min. The polyclonal anti-GFP (Molecular Probes, Inc.) and monoclonal anti-CaM (Upstate Biotechnology) antibodies were revealed using Alexa 488 and Alexa 546labeled secondary antibodies, and neurons were imaged using a PASCAL 5 laser scanning confocal microscope (Carl Zeiss MicroImaging, Inc.).
Image processing
See online supplemental material.
Pharmacological treatments
Neurons were stimulated with 50 µM glutamate or NMDA in EB containing 5 µM glycine. Membrane depolarization was triggered by 100 µM KCl for 3 min. 50 µM AP-5 (Sigma-Aldrich) was used to block the NMDA-R. The CaM antagonist w-7 (Sigma-Aldrich) was used at a 30-µM final concentration for 10 min.
CaM binding assay
PNS were prepared from HeLa cells expressing lipid-modified fluorescent probes. Cells were homogenized in 250 mM sucrose, 3 mM imidazole (pH 7.4) by passage through a 22G injection needle, and a PNS was obtained after a 10-min spin at 2,500 rpm. PNS were then incubated with CaM sepharose 4B beads (GE Healthcare) for 1 h at 4°C, in 50 mM Tris-HCl, pH 6.8, 50 mM NaCl, and 0.5% Triton X-100, in the presence of 1.3 mM Ca or 2 mM EGTA. Unbound material was removed by a quick centrifugation step. Beads were washed twice, and bound material was eluted by boiling beads in sample buffer. Input and flow-through fractions were precipitated with CHCl3/MeOH and analyzed together with the beads by SDS-PAGE (12.5%), transferred onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories), and immunoblotted using an anti-GFP antibody (Molecular Probes, Inc.) or an anti-ß-tubulin mAb. The HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) was revealed with the SuperSignal chemiluminescent substrate (Pierce Chemical Co.).
Membrane dissociation assay
We used a modified version of the protocol described by Park et al. (1997). A PNS of HeLa cells, expressing CFP-KRas-tail or CFP-Rap1a-tail (20 µg of proteins) was incubated with or without 40 µg purified CaM (Immunochemicals) in 50 mM Tris-HCl, pH 7.4, 0.5 mM MgCl2, 0.5 mM CaCl2, and 1 mM DTT, for 1 h at 37°C, and was fractionated by ultracentrifugation (50,000 rpm; for 30 min at 4°C). The resulting supernatant and membrane pellet were immunoblotted using an anti-GFP antibody as described above.
Online supplemental material
Table S1 lists lipid-modified fluorescent probes used in this work. Fig. S1 shows distribution of lipid-modified probes in hippocampal neurons. Fig. S2 depicts translocation of YFP-KRas-tail in BFA-treated neurons. Fig. S3 shows additional examples of CaM translocation to the PM in KRas-tailexpressing neurons. Fig. S4 depicts additional examples showing lack of CaM translocation in HRas-tailexpressing neurons. Fig. S5 shows a comparison of endogenous CaM and exogenous CFP-CaM expression levels.
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Acknowledgments |
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M. Fivaz is a fellow of the "Fonds National Suisse pour la recherche scientifique." This work is supported by National Institutes of Health grant MH64801 (to T. Meyer).
Submitted: 27 September 2004
Accepted: 24 June 2005
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