RalA Activation at Nascent Lamellipodia of Epidermal Growth Factor ...

Molecular Biology of the Cell Vol. 15, 2549 –2557, June 2004

RalA Activation at Nascent Lamellipodia of Epidermal Growth Factor-stimulated Cos7 Cells and Migrating D □ V Madin-Darby Canine Kidney Cells□ Akiyuki Takaya, Yusuke Ohba, Kazuo Kurokawa, and Michiyuki Matsuda* Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita-shi, Osaka 565-0871, Japan Submitted November 28, 2003; Revised February 16, 2004; Accepted March 1, 2004 Monitoring Editor: Martin A. Schwartz

RalA, a member of the Ras-family GTPases, regulates various cellular functions such as filopodia formation, endocytosis, and exocytosis. On epidermal growth factor (EGF) stimulation, activated Ras recruits guanine nucleotide exchange factors (GEFs) for RalA, followed by RalA activation. By using fluorescence resonance energy transfer-based probes for RalA activity, we found that the EGF-induced RalA activation in Cos7 cells was restricted at the EGF-induced nascent lamellipodia, whereas under a similar condition both Ras activation and Ras-dependent translocation of Ral GEFs occurred more diffusely at the plasma membrane. This EGF-induced RalA activation was not observed when lamellipodial protrusion was suppressed by a dominant negative mutant of Rac1, a GTPase-activating protein for Cdc42, inhibitors of phosphatidylinositol 3-kinase, or inhibitors of actin polymerization. On the other hand, EGF-induced lamellipodial protrusion was inhibited by microinjection of the RalA-binding domains of RalBP1 and Sec5. Furthermore, we found that RalA activity was high at the lamellipodia of migrating Madin-Darby canine kidney cells and that the migration of Madin-Darby canine kidney cells was perturbed by the microinjection of RalBP1–RalA-binding domain. Thus, RalA activation is required for the induction of lamellipodia, and conversely, lamellipodial protrusion seems to be required for the RalA activation, suggesting the presence of a positive feedback loop between RalA activation and lamellipodial protrusion. Our observation also demonstrates that the spatial regulation of RalA is conducted by a mechanism distinct from the temporal regulation conducted by Ras-dependent plasma membrane recruitment of Ral guanine nucleotide exchange factors.

INTRODUCTION RalA and RalB are members of the Ras-family GTPases (Chardin and Tavitian, 1986; Chardin and Tavitian, 1989) and reside both at the plasma membrane and endomembrane compartments (Feig et al., 1996). RalA and RalB, collectively called Ral hereafter, are 85% identical in their amino acid sequences, and their differences are mostly in their carboxy-terminal hypervariable regions (Chardin and Tavitian, 1989). Similarly to the other Ras-family G proteins, Ral is regulated primarily by two classes of proteins, Ralspecific guanine nucleotide exchange factor (Ral GEFs) and Ral GTPase-activating protein(s) (Ral GAP) (Feig et al., 1996; Quilliam et al., 2002); however, the molecular identity of the latter remains unknown. Article published online ahead of print. Mol. Biol. Cell 10.1091/ mbc.E03–11– 0857. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E03–11– 0857. □ D □ V Online version of this article contains supporting material. Online version is available at www.molbiolcell.org. * Corresponding author. E-mail address: matsudam@biken. osaka-u.ac.jp. Abbreviations used: EGF, epidermal growth factor; FITC, fluorescein isothiocyanate; FRET, fluorescence resonance energy transfer; GAP, GTPase activating protein; GEF, guanine nucleotide exchange factor; GFP, green fluorescence protein; PDK1, phosphatidylinositol 3-kinase– dependent kinase 1; PI-3K, phosphatidylinositol 3-kinase; Raichu, Ras and interacting protein chimeric unit.

© 2004 by The American Society for Cell Biology

Many Ral GEFs, such as RalGDS, Rlf/Rgl2, Rgl, Rgl3/ RPM, and Rgr, contain a Ras-binding domain and are activated by binding to Ras (for review, see Feig et al., 1996; Bos, 1998; Wolthuis and Bos, 1999; Quilliam et al., 2002). Thus, Ral proteins become activated in response to the growthstimulating ligands, including epidermal growth factor (EGF), that activate Ras. Consequently, Ral mediates some of the cellular actions of Ras, particularly those related to the tumorigenic activity of Ras (Wolthuis et al., 1998b; Ward et al., 2001; Takai et al., 2001). Recently, it has been suggested that this Ral GEF pathway plays a more important role than do phosphatidylinositol 3-kinase (PI-3K) and Raf in the Rasdependent oncogenesis in human cells (Hamad et al., 2002). Notably, the Ras-binding and resultant membrane recruitment of Ral GEFs may not be sufficient for the activation of Ral. For example, another Ras-family G protein, Rap1A, cannot activate Ral irrespective of its high-affinity binding to some Ral GEFs (Urano et al., 1996). Requirement of phosphatidylinositol 3-dependent kinase 1 (PDK1), a downstream effector of PI-3K, has been proposed to account for this discrepancy (Tian et al., 2002). Furthermore, ␤-arrestin and protein kinase C also regulate the activity of RalGDS (Rusanescu et al., 2001; Bhattacharya et al., 2002). In addition to this Ras-dependent pathway, increased levels of intracellular calcium are known to increase GTP loading of Ral (Hofer et al., 1998; Wolthuis et al., 1998a). Unlike in the case of Ras, however, increased levels of calcium may directly activate Ral by the binding of the calcium-binding protein calmodulin to the carboxyl-terminal region of Ral (Wang and Roufogalis, 1999).

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Ral exerts its effects via various Ral-binding proteins. Among them, Ral binding protein 1 (RalBP1)/Ral interacting protein (RIP/RLIP) is the first Ral effector to be discovered (Cantor et al., 1995; Jullien-Flores et al., 1995; Park and Weinberg, 1995). RalBP1 is proposed to regulate endocytosis of EGF receptor, insulin receptor, and transferrin receptor via POB1 (Nakashima et al., 1999) and ␮2, the medium chain of AP2 complex (Jullien-Flores et al., 2000). On the other hand, Ral may also regulate exocytosis and filopodia formation by its binding to Sec5 and Exo84, constituents of the exocyst complex (Moskalenko et al., 2002; Sugihara et al., 2002; Moskalenko et al., 2003). Another Ral effector that is implicated in vesicular trafficking is phospholipase D1; however, phospholipase D1 is not a typical effector in that it binds constitutively to the amino-terminal region of Ral (Jiang et al., 1995). Fluorescence resonance energy transfer (FRET) is a quantum-mechanical phenomenon that occurs between two fluorophores, called the donor and acceptor. When the donor is in molecular proximity to the acceptor, and when the emission spectrum of the donor overlaps the excitation spectrum of the acceptor, the energy of the donor is transferred to the acceptor, often resulting in emission from the acceptor. Variants of green fluorescent protein (GFP) have been shown to provide genetically encoded fluorophores that serve as donor and/or acceptor in FRET (Miyawaki and Tsien, 2000). By using these GFP variants and FRET, several intracellular signaling events have been successfully visualized in a single living cell (Tsien and Miyawaki, 1998; van Roessel and Brand, 2002). We have also reported probes, called Raichu (Ras and interacting protein chimeric unit) probes, for the monitoring of the activities of Ras, Rap1, Rac, Cdc42, and RhoA. (Mochizuki et al., 2001; Itoh et al., 2002; Yoshizaki et al., 2003). Here, we report on the development of a series of probes for RalA activity, which we exemplify by a probe designated Raichu-RalA, and show that RalA is activated at nascent lamellipodia in EGF-stimulated Cos7 cells and migrating Madin-Darby canine kidney (MDCK) cells. MATERIALS AND METHODS Plasmids pRaichu-Ras have been described previously (Mochizuki et al., 2001). The expression plasmid pRaichu-RalA encoding the probe named Raichu-RalA was constructed essentially as pRaichu-Ras. From the amino terminus, Raichu-RalA consists of yellow fluorescent protein (YFP) (aa 1–239), spacer (Leu-Asp), rat RalA (aa 12–188), spacer (Ser-Gly-Gly-Thr-Gly-Gly-Gly-GlyThr), the RalA-binding domain (RBD) of RalBP1 (aa 423– 489), spacer (GlyGly-Arg), cyan fluorescent protein (CFP) (aa 1–237), spacer (Gly-Arg-SerArg), and the carboxy-terminal region of RalA (aa 183–206) (Figure 1A). In this study, we used YFP [Phe 47 Ala, Thr 66 Gly, Val 69 Leu, Ser 73 Ala, Met 154 Thr, Val 164 Ala, Ser 176 Gly, and Thr 204 Tyr] and CFP [Lys 27 Arg, Tyr 67 Trp, Asp 130 Ala, Asn 147 Ile, Met 154 Thr, Val 164 Ala, Asn 165 His, and Ser 176 Gly] as the acceptor and donor, respectively. In proteins denoted by suffices -V23 and -N28, Gly23 and Thr28 of RalA were replaced with Val and Asn, respectively. Synthesized cDNA of a fluorescent protein dsFP593 was obtained from A. Miyawaki (Brain Science Institute, RIKEN, Wako-shi, Japan). Raichu-RalA/K-RasCT and Raichu-RalA/Rap1CT contained the carboxy-terminal region of K-Ras (aa 169 –188) and Rap1 (aa 168 –184). pIRM21 was an expression vector derived from pCAGGS (Niwa et al., 1991) and contained the internal ribosomal entry site and the coding region of dsFP593 (Fradkov et al., 2000) at the 3⬘ region of the multiple cloning site. pIRM-FlagRac-N17 was constructed by inserting the cDNA of a dominant negative Rac1, Rac1-N17, into pIRM21(Mochizuki et al., 2000). In a similar manner, we constructed pIRM-Flag-H-Ras, pIRM-Flag-K-Ras, pIRM-Flag-H-Ras-N17, pIRM-Flag-Rap1A, pIRM-KIAA0362, pIRM-p115RhoGEF, pIRM-Gap1m, pIRM-rap1GAPII, pIRM-KIAA0053, pIRM-KIAA1204, and pIRM-Flagp50RhoGEF. pDsRed2-RalA is constructed from pDsRed2-C1 vector (BD Biosciences Clontech, Palo Alto, CA). pCAGGS-mSos1 and –myc-C3G were constructed from pCAGGS. An expression vector for glutathione S-transferase (GST)-RalBP1-RBD and pcDNA3-Rlf was obtained from J.L. Bos (Utrecht University, Utrecht, The Netherlands). pcDNA3-RalGDS was obtained from

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Figure 1. Basic properties of Raichu-RalA. (A) Schematic representation of Raichu-RalA bound to GDP or GTP. Ral, RBD, GEF, and GAP indicate RalA, the Ral-binding domain of RalBP1, the guanine nucleotide exchange factor for Ral, and the Ral GTPase-activating protein, respectively. YFP and CFP denote yellow-emitting and cyan-emitting mutants of GFP, respectively. (B) Emission spectra of Raichu-RalA expressed in 293T cells. Cells were lysed and analyzed with a fluorescence spectrometer at an excitation wavelength of 433 nm. WT, V23, and N28 denote the wild-type, constitutively active mutant, and dominant negative mutant, respectively. (C) GTP/GDP loading of Raichu-RalA and DsRed2-tagged RalA. 293T cells expressing Raichu-RalA or DsRed2-RalA were labeled with 32Pi. Raichu-RalA and DsRed2-RalA were precipitated with anti-GFP and anti-RFP rabbit sera, respectively, followed by TLC analysis. Separated GTP and GDP were quantitated with a BAS-1000 image analyzer and GTP/(GTP ⫹ GDP) (%) values were plotted with SD.

A. Wittinghofer at Max-Plank Institute. pCAGGS-EGFP-Rlf and -RalGDS were constructed from pCAGGS-EGFP (Ohba et al., 2000) and pcDNA3-Rlf or -RalGDS. cDNA of Sec5 was obtained from Y. Ohta (Harvard Medical School, Cambridge, MA). A DNA fragment corresponding to aa 1–99 of Sec5 was polymerase chain reaction-amplified and subcloned into pGEX-4T3.

Cells, Antibodies, and Reagents Cos7 cells used in this study were Cos7/E3, a subclone of Cos7 cells established by Y. Fukui (University of Tokyo, Tokyo, Japan). 293T cells were a gift from B.J. Mayer (University of Connecticut, Storr, CT). MDCK cells were purchased from the Human Science Research Resources Bank (Sennan-shi, Osaka, Japan). Cells were maintained in DMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal calf serum. Anti-Akt and anti-phosphoAkt (Thr308) were purchased from Cell Signaling Technology (Beverly, MA). Anti-green fluorescent protein (GFP) rabbit serum was prepared in our laboratory. Anti-red fluorescent protein (RFP) polyclonal antibody was a gift from N. Mochizuki at the National Cardiovascular Center (Suita-shi, Osaka, Japan). Anti-RalA was purchased from BD Transduction Laboratories (Lexington, KY). Cytochalasin D and latrunculin B were purchased from Calbiochem (San Diego, CA). EGF, wortmannin, and LY294002 were purchased from Sigma-Aldrich.

Molecular Biology of the Cell

RalA Activation at Nascent Lamellipodia

In Vitro Spectroscopy Spectrograms of cells were obtained essentially as described previously (Mochizuki et al., 2001). Plasmids were transfected into 293T cells by the calcium phosphate coprecipitation method. Thirty-six hours later, cells were harvested in lysis buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% Triton X-100, 5 mM MgCl2) and clarified by centrifugation. The fluorescence spectrum was obtained by use of an excitation wavelength of 433 nm with an FP-750 spectrofluorometer (Jasco, Tokyo, Japan).

Analysis of Guanine Nucleotides Bound to G Proteins Guanine nucleotides bound to Raichu probes and DsRed2-RalA were analyzed essentially as described previously (Matsuda et al., 1994). Briefly, 293T cells were transfected with pRaichu-RalA or pDsRed2-RalA. Thirty-six hours after transfection, cells were labeled with 32Pi in phosphate-free modified Eagle’s medium (Invitrogen, Carlsbad, CA) for 4 h. Raichu-RalA and DsRed2RalA were immunoprecipitated with anti-GFP antibody and anti-RFP antibody, respectively. The immunoprecipitates were boiled and analyzed by thin layer chromatography (TLC). The amount of GTP and GDP bound to RalA was quantitated with a BAS-1000 image analyzer (Fuji Film, Tokyo, Japan).

Bos’ Pull-Down Assay Bos’ pull-down assay was performed essentially as described previously (Wolthuis et al., 1998b). Cos7 cells either pretreated for 30 min with 500 nM wortmannin, 20 ␮M LY294002, 2 ␮M cytochalasin D, and 2 ␮M latrunculin B, or not pretreated were washed twice by Tris-buffered saline containing 1 mM Na3VO4, lysed in Ral buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2.5 mM MgCl2, 1% NP-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 ␮g/ml aprotinin, and 10 ␮g/ml leupeptin), and clarified by centrifugation. The supernatant was incubated with GST-RalBP1-RBD fusion proteins. The resulting complexes of GTP-bound Ral and GST-RBD were precipitated with glutathione-Sepharose beads (Amersham Biosciences, Piscataway, NJ) and separated by SDS-PAGE, followed by immunoblotting with anti-Ral antibody. Bound antibodies were detected by an enhanced chemiluminescence detection system (Amersham Biosciences) and quantitated with an LAS-1000 image analyzer (Fuji Film).

Immunoblotting Cos7 cells were washed twice with phosphate-buffered saline, lysed in lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl2, 1% NP-40, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 ␮g/ml aprotinin, and 10 ␮g/ml leupeptin), and clarified by centrifugation. The supernatant was analyzed by SDS-PAGE, followed by immunoblotting.

Imaging of RalA Activity in Living Cells Stimulated with EGF RalA activity was imaged with Raichu-RalA probe essentially as described previously (Mochizuki et al., 2001). Expression plasmids were transfected into Cos7 cells by Polyfect (QIAGEN, Valencia, CA). After 24 h, cells were serum starved for 2 h. Then, the cells were imaged at 1-min intervals on an Axiovert 200 inverted microscope (Carl Zeiss, Jena, Germany) that was equipped with a cooled charge-coupled device (CCD) camera, CoolSNAP HQ (Roper Scientific, Trenton, NJ), and controlled by MetaMorph software (Universal Imaging, West Chester, PA) (Miyawaki et al., 1997; Mochizuki et al., 2001). For dual-emission ratio imaging of Raichu probes, we used a 455DRLP dichroic mirror (Omega Optical, Brattleboro, VT), 86436 excitation filter, and two emission filters, 86470 for cyan fluorescent protein (CFP) and 86535 for YFP (Chroma Technology, Brattleboro, VT). Cells were illuminated with a 75-W xenon lamp and a 63⫻ oil immersion objective lens. The exposure time was 0.1 s for YFP and 0.2 s for CFP when the binning of the CCD camera was set to 4 ⫻ 4. Starting from 10 min, cells were stimulated with 50 ng/ml EGF. After background subtraction, the ratio image of YFP/CFP was created with the MetaMorph software and used to represent FRET efficiency. In some experiments, GST-fusion proteins were microinjected into the cells 30 min before the analysis.

Confocal Microscopy Cos7 cells plated on glass base dishes were imaged with an FV-500 confocal microscope (Olympus, Tokyo, Japan) equipped with an Argon laser as described previously (Ohba et al., 2000). After acquiring 20 XY images scanned from the bottom to the top of the cells, stacked images for XY sections and XZ sections were obtained.

Wound Healing Assay MDCK cells were seeded at a high density on 30-mm glass bottom dishes. Expression plasmids of Raichu probes were transfected into the cells with LipofectAMINE 2000 (Invitrogen). When the cells formed a confluent monolayer, they were wounded by scraping with a microinjection needle, rinsed with phosphate-buffered saline, and fed with fresh medium containing 10% serum. Beginning at 1 h after wounding, cells were imaged every 2 min for

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4 h. In some experiments, cells at the edge of the wound were microinjected with fluorescein isothiocyanate (FITC)-dextran and GST-fusion proteins (1 mg/ml) 30 min before the time-lapse analysis and imaged for FITC and phase contrast (PC) for 4 h.

Microinjection GST, GST-RalBP1-RBD, GST-Sec5-RBD, or GST-PAK-CRIB at a concentration of 1 mg/ml was microinjected into Cos7 or MDCK cells with a set of manipulators (micromanipulator 5171 and FemtoJet; Eppendorf, Hamburg, Germany). As a marker for the microinjected cell, 5 mg/ml FITC-coupled dextran (Sigma-Aldrich) was coinjected with the fusion proteins.

RESULTS Development of Raichu-RalA For the visualization of RalA activity in living cells, we constructed and tested a series of probes, where the order of RalA and RBD was changed or the amino- and carboxyterminal regions of RalA and RBD were trimmed to various lengths. For the sake of brevity, we describe only the probe named Raichu-RalA, which showed the best signal-to-noise ratio and dynamic range. From the amino terminus, RaichuRalA consists of a modified YFP called Venus, rat RalA (aa 12–188), RBD of RalBP1 (aa 423– 489), and a modified CFP called SECFP, followed by the carboxy-terminal hypervariable region of RalA (aa 183–206) (Figure 1A). Spacer sequences consisting of two to nine amino acids were intercalated between these domains. In this probe, the intramolecular binding of GTP-RalA to RBD is expected to bring CFP close to YFP, thereby increasing FRET from CFP to YFP. To examine whether GTP loading increased FRET efficiency, we prepared Raichu-RalA-V23 and Raichu-RalAN28. In Raichu-RalA-V23, Gly23 was replaced with valine to reduce the sensitivity to GAP, thereby increasing the GTP loading of this mutant. In Raichu-RalA-N28, Thr28 was replaced with Asn, a mutation that is known to reduce the affinity of G proteins to guanine nucleotides. The probes were expressed in 293T cells, and their fluorescence emission profiles were obtained by the use of an excitation wavelength of 433 nm (Figure 1B). Because FRET was most typically observed as an increase in the emission peak of 527 nm and a concurrent decrease in the emission peak of 475 nm, the emission ratio of 527 nm versus 475 nm is used to represent the FRET efficiency hereafter. As shown in Figure 1B, the emission ratio of wild-type Raichu-RalA was lower than that of the V23 mutant and higher than that of the N28 mutant. This result indicated that the emission ratio of Raichu-RalA was correlated with the GTP/GDP ratio on the probes. Correlation of the GTP Loading of the Probes with That of the Authentic G Proteins To confirm the correlation between the GTP/GDP loading of the probes and that of the authentic RalA, we quantitated the guanine nucleotides bound to Raichu-RalA and DsRed2tagged RalA by TLC (Figure 1C). The GTP level of RaichuRalA was 13.3 ⫾ 2.0% and that of DsRed2-RalA was 7.0 ⫾ 2.0%. The GTP levels on Raichu-RalA-V23 and DsRed2RalA-V23 were 58.4 ⫾ 4.6 and 54.1 ⫾ 9.1%, respectively. In the N28 mutants, the binding of guanine nucleotides was markedly reduced, which reflected the weak affinity to the guanine nucleotides in these mutants. These results indicated that the GTP/GDP level on the probe paralleled that on the authentic RalA, and probably that on the endogenous RalA. Correlation of FRET Efficiency with GTP Loading To examine the correlation between GTP loading of the probe and the FRET efficiency, a dose-responsive curve was

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Figure 2. Sensitivity of Raichu-RalA to GEFs and GAPs. (A) Correlation of GTP-loading and FRET efficiency of Raichu-RalA. pRaichu-RalA and varying quantities of pcDNA3-Rlf were transfected into pairs of 293T cells. One set of cells was labeled with 32Pi and determined for GTP/(GTP ⫹ GDP) (%) on Raichu-RalA by TLC. The other set of cells was lysed and examined for the emission ratio (intensity at 475 vs. 530 nm) with an excitation wavelength of 433 nm with a fluorescence spectrometer. Error bars are shown either for the upward or the downward for the brevity. (B) FRET efficiency of Raichu-RalA in the presence of various GEFs and GAPs. pRaichu-RalA was transfected into 293T cells with expression vectors encoding GEFs or GAPs as indicated at the bottom of the figure. FRET efficiency was measured as in A. Data from at least two independent experiments are shown with SD.

obtained with various quantities of an Rlf expression plasmid. As shown in Figure 2A, both the GTP loading and FRET efficiency of the probe increased with the expression of Rlf in a dose-dependent manner. The FRET efficiency and GTP loading showed a near-linear correlation over a wide GTP range. We also examined the specificity of the probes by using GEFs and GAPs for the Ras superfamily G proteins. As shown in Figure 2B, Rlf and RalGDS increased the FRET efficiency remarkably. In contrast, mSos1, C3G, KIAA0362, and p115RhoGEF, which are GEFs for Ras, Rap1, Rac, and Rho, respectively, could not increase the FRET efficiency of Raichu-RalA to a detectable level. Because Ral GAP has not been identified, we could not examine the sensitivity of Raichu-RalA to RalGAP in detail. However, we could show the ineffectiveness of the expression of Gap1m, rap1GAPII, KIAA0053, and p50RhoGAP, which are GAPs for Ras, Rap1, Rac, and Rho, respectively. These results demonstrated the versatility of Raichu-RalA for the monitoring of the balance between the GEF and GAP activities of RalA. Because this balance between GEF and GAP primarily determines the GTP/GDP ratio on RalA, we will call this activity balance the RalA activity hereafter.

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Figure 3. RalA activity in EGF-stimulated Cos7 cells. Cos7 cells were transfected with pRaichu-RalA or pRaichu-Ras. Cells were imaged for YFP, CFP, and phase contrast (PC) every 1 min with a time-lapse epifluorescence microscope equipped with a cooled CCD camera. The ratio images (YFP/CFP) were generated with MetaMorph software. In the IMD mode shown here, eight colors from red to blue are used to represent the YFP/CFP ratio, with the intensity of each color indicating the mean intensity of YFP and CFP. The upper and lower limits of the ratio range are shown on the right. (A) Representative ratio images of Raichu-RalA and RaidhuRas at the indicated time points. EGF (50 ng/ml) was inoculated at 0 min. Cell imaging was repeated ⬎20 times, and the most representative images are shown. (B) Ratio and phase contrast images of cells expressing Raichu-RalA at 10 min after EGF stimulation. White broken lines show the contours of the cells before EGF stimulation. Bars, 25 ␮m.

Imaging of RalA Activity in Cos7 Cells upon EGF Stimulation Using Raichu-RalA, we visualized RalA activity in living Cos7 cells. To demonstrate the local RalA activity in living cells, the FRET images were displayed in the intensity-modulated display mode (Figure 3, A and B, and Fig 3RalA.mov). Basal RalA activity was higher at the peripheral plasma membrane than at the central region of the serum-starved Cos7 cells. On stimulation with EGF, the RalA activity was rapidly increased at the nascent lamellipodia. We observed similar pattern of RalA activation by using Raichu-RalA probes fused to the carboxy terminus of K-Ras4B or Rap1A (Supplementary Figure 1). Because Raichu-RalA/K-Ras4B-CT localizes mostly at the plasma membrane and Raichu-RalA/Rap1A-CT localizes both endomembrane compartments and the plasma membrane, the difference in the probe distribution did not seem to bias the



detectable regions of RalA activation toward lamellipodia. In cells expressing Raichu-RalA-S28N, EGF stimulation induced lamellipodial protrusion but did not increase in FRET efficiency. To understand the activation mechanism of RalA, we compared the activation pattern of RalA with that of Ras (Figure 3A and Fig 3Ras.mov). On EGF stimulation, Ras was rapidly activated from the periphery of the cells. Importantly, however, Ras activation was not limited to the lamellipodial protrusion. Expression of a dominant negative mutant of HRas, H-Ras-N17, completely inhibited RalA activation (our unpublished data). These observations suggested that EGF-induced RalA activation was mediated by Ras, but the local Ras activation was not sufficient for the RalA activation. Ral GEFs Recruitment to Plasma Membrane upon EGF Stimulation Why is RalA activated only at the nascent lamellipodia, whereas Ras is activated more diffusely at the plasma membrane? One possible explanation is that Ral GEFs are recruited only to the lamellipodial protrusion. To investigate this possibility, we examined the EGF-induced translocation of GFP-tagged Ral GEFs Rlf and RalGDS. For the quantitative visualization of GFP-Rlf and RalGDS recruitment to Ras, H-Ras or K-Ras was coexpressed in Cos7 cells (Figure 4). Before stimulation, the fluorescence of GFP-Rlf showed a typical cytosolic pattern. Five minutes after EGF stimulation, GFP-Rlf coexpressed with H-Ras or K-Ras was dispersed diffusely throughout the entire cell, suggesting its translocation from the cytoplasm to the plasma membrane (Figure 4A). However, we did not observe any accumulation of GFP-Rlf at the nascent lamellipodia. Very similar results were obtained by using GFP-RalGDS (Figure 4B and Fig 4RalGDS.mov). To quantitate the plasma membrane translocation, we measured the intensities of two regions defined at the perinuclear area and at the peripheral area, respectively. Then, the ratio of the latter to the former was used as the index of membrane recruitment. This method is based on the assumption that the intensity at the perinuclear region reflects the fluorescence from the cytosolic GFP-Rlf and GFP-RalGDS than does the intensity at the peripheral region. The time courses of the EGF-induced redistribution of GFP-Rlf and GFP-RalGDS were essentially the same between the cells expressing H-Ras and K-Ras (Figure 4C). We also performed similar experiments with a minimum detectable level of GFP-RalGDS in the absence of exogenous Ras. Although the level of translocation was not as remarkable as in the presence of exogenous H-Ras, similar results were obtained in the absence of exogenous H-Ras (Supplementary Figure 2A). Furthermore, we examined whether the localized RalA activation occurred in the condition where diffuse translocation of Ral GEFs was observed in the presence of exogenous Ras and Ral GEFs. Albeit coexpression of RalGDS with Raichu-RalA increased the basal level of RalA activity and rendered the EGF-induced increase less remarkable, the EGF-induced RalA activation was observed mostly at nascent lamellipodia (Supplementary Figure 2B). These observations revealed the following: 1) Ral GEFs are translocated to the activated Ras at the plasma membrane, and their distribution is not limited to the nascent lamellipodia; and 2) Ral GEFs recruitment to the activated Ras is not sufficient for the activation of RalA. Requirement of PI-3K and Rac/Cdc42 Activation for the EGF-induced RalA Activation During the course of our study to visualize the activity of low-molecular-weight GTPases, we noticed that the localization of the activated RalA resembled the localizations of

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Figure 4. Translocation of RalGEFs to the plasma membrane upon EGF stimulation. (A and B) pCAGGS-EGFP-Rlf or -RalGDS was cotransfected with pIRM-Flag-H-Ras or pIRM-Flag-K-Ras into Cos7 cells. After 24 h, cells were serum starved for 4 h and stimulated with 50 ng/ml EGF. Expression of the GTPases was confirmed by the fluorescence of dsFP593, which was translated from the internal ribosomal entry site of pIRM. Images of EGFP-Rlf and -RalGDS were obtained every 1 min, and EGF was added 5 min after the start of recording. The images of H-Ras and K-Ras were taken are at 0 and 15 min. (C) Intensity ratios of the regions denoted by circles A and B in A and B were plotted against time. The intensity ratio was defined as IB/IA, where IA is the intensity of perinuclear region A, and IB is the intensity of peripheral region B. Bars, 25 ␮m.

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cultured cells (Aoki et al., 2004). We have recently shown that expression of CdGAP inhibits EGF-induced lamellipodial protrusion without perturbing Rac1 activation (Kurokawa et al., 2004). In the presence of CdGAP, EGF-induced RalA activation was significantly suppressed (Figure 5, A and B). Furthermore, we examined the effect of PI-3K inhibitors, which are known to perturb EGF-induced lamellipodial formation and Rac1 activation in Cos7 cells (Shinohara et al., 2002). As shown in Figure 5, C and D, both wortmannin and LY294002 inhibited the EGF-induced RalA activation, demonstrating the requirement of PI-3K for RalA activation. Because each of Rac1, Cdc42, and PI-3K was required for the EGF-induced lamellipodial protrusion in Cos7 cells, we speculated that nascent lamellipodia per se were required for the activation of RalA. To examine this possibility, we treated the cells with inhibitors of actin polymerization and stimulated with EGF. In support of our hypothesis, both cytochalasin D and latrunculin B completely inhibited EGFinduced RalA activation in Cos7 cells. Importantly, EGFinduced activation of Rac and Ras was not inhibited by these PI-3K inhibitors in Cos7 (our unpublished data). On the other hand, expression of the dominant negative mutant of Rac1, CdGAP, or treatment with the inhibitors of actin polymerization did not significantly inhibit EGF-induced activation of PI-3K as examined by the phosphorylation of Akt (Figure 5E). Interestingly, these inhibitors of actin polymerization inhibited EGF-induced activation of Cdc42, but not of Rac1 (Supplementary Figure 3). These results strongly suggested that EGF-induced RalA activation required not only Ras but also PI-3K and Rac/Cdc42, probably because actin remodeling at the lamellipodia played a critical role in the activation of RalA.

Figure 5. Inhibition of EGF-induced RalA activation by reagents that perturb lamellipodia. (A) Cos7 cells transfected with or without an expression vector for a dominant negative mutant of Rac, Rac1-N17, or GAP for Cdc42, CdGAP were serum starved for 8 h and stimulated with EGF. Cells were analyzed by Bos’ pull-down method. (B) Levels of GTP-RalA were quantitated with an LAS-1000 image analyzer. Data from two independent experiments are shown with SD. (C and D) Cos7 cells left untreated or treated with wortmannin (Wort), LY294002 (LY), latrunculin B (LatB), or cytochalasin D (CytD) for 30 min were stimulated with EGF and analyzed as described in A. Data from three independent experiments are shown with SD. (E) Cells treated as in A and C were analyzed for the phosphorylation of Akt with anti-phospho-Akt (Thr308) antibody.

activated Rac1 and Cdc42 (Kurokawa et al., 2004), which urged us to investigate the requirement of Rac1 and Cdc42 for the RalA activation. First, we transfected Cos7 cells with Raichu-RalA and a dominant negative mutant of Rac1, Rac1N17, and monitored the activity change of RalA upon EGF stimulation. The expression of Rac1-N17 suppressed both the induction of lamellipodia and RalA activation as observed with Raichu-RalA (our unpublished data). Next, this observation was confirmed by Bos’ pull-down assay. As shown in Figure 5, A and B, the EGF-dependent increase in GTP-RalA was completely inhibited by the expression of Rac1-N17. Because Cdc42-N17, a dominant negative Cdc42 mutant, may sequester not only Cdc42 GEFs but also Rac GEFs, we used CdGAP, which is a GAP specific for Cdc42 in

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Requirement for Ral in the Lamellipodial Protrusion What is the role of Ral activation at the nascent lamellipodia? To investigate this, we perturbed Ral signaling by the microinjection of GST-tagged Ral-binding domains of RalBP1 and Sec5. As a control, we also microinjected GST alone or GST-PAK-CRIB, which is known to block the Rac1 and Cdc42 pathways. The phenotype of EGF-stimulated Cos7 cells consisted of two distinct morphological changes, lamellipodial protrusion and membrane ruffling. We quantitated the lamellipodial protrusion by measuring the length of outward extension of the plasma membrane (Figure 6A). Although the level of inhibition was slightly different, both GST-RalBP1-RBD and GST-Sec5-RBD inhibited EGF-induced lamellipodia protrusion (Figure 6B), demonstrating the essential role of Ral in this process. Notably, in Cos7 cells microinjected with GST-RalBP1-RBD or GST-Sec5-RBD, membrane ruffling was not significantly suppressed, whereas both lamellipodial protrusion and membrane ruffling were inhibited in cells microinjected with GST-PAK-CRIB. Effect of RalA Inhibition on the Migration of MDCK Cells To investigate the role of RalA activation at lamellipodia in a different biological context, we imaged the RalA activity during the wound healing process of MDCK cells (Figure 7 and Fig 7RalA.mov). Activity of RalA was high in the lamellipodia that protruded toward the direction of cell migration. Again, the distributions of active Rac1 and Cdc42 were very similar to that of RalA; however, Ras did not show any spatial gradient in its activity within the migrating MDCK cells (Figure 7 and Fig 7Cdc.mov). Next, we microinjected GST, GST-RalBP1-RBD, or GSTPAK-CRIB into the MDCK cells at the edge of the wound (Figure 8A). The velocities of migrating MDCK cells were slowed in the presence of either GST-RalBP1-RBD or GST-



Figure 6. Inhibition of EGF-induced lamellipodial protrusion by RalBP1-RBD and Sec5-RBD. GST, GST-RalBP1-RBD, Sec5-RBD, or GST-PAK-CRIB were microinjected into Cos7 cells. Beginning at 30 min, phase contrast images of these cells were obtained every 30 s, followed by the stimulation with EGF (50 ng/ml). Cells images were further collected for 30 min. (A) Outline of the quantification of lamellipodial induction. The maximum length of nascent lamellipodia (X) and the breadth of the nucleus (Y) were measured on the video image of each microinjected cell. When the value of X/Y was ⬎0.3, the cell was scored as lamellipodia positive. (B) More than 60 cells were analyzed for each protein in at least six independent experiments, and the percentage of lamellipodia-positive cells is shown.

PAK-CRIB (Figure 8B), indicating that the high RalA activity at the lamellipodia was required for the migration of MDCK cells. This observation confirmed that RalA plays an essential role at the lamellipodial protrusion. DISCUSSION The clear difference in the localization of Ras and RalA activities provides a clue to the understanding of the spatial regulation of RalA activity. Both Ras activation and Ral GEFs recruitment occurred more diffusely at the plasma membrane (Figures 3 and 4). Although it has been reported that H-Ras, but not K-Ras, activates Ral (Xu et al., 2003), we have found that both H-Ras and K-Ras served as plasma membrane docking sites for Rlf and RalGDS (Figure 4). These observations indicate that plasma membrane recruitment of Ral GEFs is not sufficient for RalA activation, implying the presence of another activator(s) of Ral GEFs that is stimulated by H-Ras, but not K-Ras. PDK1 fulfills these criteria. First, PDK1 binding to RalGDS stimulates the GEF activity of RalGDS (Tian et al., 2002). Second, H-Ras is known to be a more potent stimulator of PI-3K than K-Ras (Yan et al., 1998). However, translocation of PDK1 to the plasma membrane is not limited to the lamellipodial protrusion (Anderson et al., 1998; Filippa et al., 2000), and the PH domain of PDK1 is not required for RalGDS activation (Tian et al., 2002). Thus, PDK1 recruitment to the plasma membrane does not seem to explain the discrepancy between the activation sites of Ras and RalA. A clue for understanding the spatial regulation of RalA may reside in the observation that RalA was not activated in the absence of nascent lamellipodia, i.e., a dominant negative mutant of Rac1, a GAP for Cdc42, and inhibitors of PI-3K or actin polymerization perturbed both lamellipodial protrusion and RalA activation in the EGF-stimulated Cos7 cells. We have shown that the intracellular nonuniformity of GAP activity is primarily responsible for the difference in the activation sites between Ras and Rap1 in EGF-stimulated cells (Ohba et al.,

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Figure 7. Imaging of activities of low-molecular-weight GTPases in migrating MDCK cells. Confluent MDCK cells transfected with an expression plasmid for Raichu-RalA, Raichu-Ras, Raichu-Rac1, or Raichu-Cdc42 were wounded 1 h before FRET imaging. Images of CFP, YFP, and DIC were obtained every 2 min for 4 h. Shown here are representative pseudocolor images of FRET efficiency (YFP/CFP) and overlay images of DIC and YFP at 2 h from the start of FRET imaging. Ratio ranges are shown on the right. Each cell imaging was repeated at least three times with similar results. Bars, 25 ␮m.

2003); therefore, a similar mechanism may account for the spatial regulation of RalA. Partial purification of proteins with Ral GAP activity has been reported (Emkey et al., 1991; Bhullar and Seneviratne, 1996), but no group has successfully identified and cloned the Ral GAP gene. Without knowing the molecular identity of Ral GAP(s), it is difficult to foretell the mechanism of spatial regulation by Ral GAP at this time. Nevertheless, we may be able to speculate that Ral GAP is present in the cytosol, but it cannot reach to RalA at the extending lamellipodia due to the dense actin meshwork sandwiched with the dorsal and basal plasma membranes. When the RalA pathway was abrogated by the microinjection of Ral binding domain of RalBP1 or Sec5, both the EGFinduced lamellipodial protrusion of Cos7 cells and migration of MDCK cells were perturbed (Figures 6 and 8). These observations agree with previous reports demonstrating the requirement of RalA for the membrane ruffling in N-formyl-l-methionyl-l-leucyl-l-phenylalanine–stimulated human embryonic kidney 293 cells (Bhattacharya et al., 2002) and for the migration of myoblasts and T24 bladder cancer cells (Suzuki et al., 2000; Gildea et al., 2002; Neuhaus et al., 2003). Then, how does RalA activation at the lamellipodia contribute to the lamellipodial protrusion and cell migration? It has been reported that the surfaces of membrane ruffles arise by exocytosis of the internal

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creased by trimming of both the amino and carboxy termini. However, this deletion deprived the probe of its sensitivity to phospholipase D1 and calmodulin, which are known to bind to the N terminus and the C terminus of RalA, respectively (Jiang et al., 1995; Wang et al., 1997). Furthermore, the carboxy-terminal region (aa 178 –206) of RalA has been shown to be necessary for the activation by RalGEF2/ RalGPS1B (de Bruyn et al., 2000). In contrast to other Ral GEFs, RalGEF2/RalGPS1B lacks the Ras-binding domain, but it contains a Grb2-binding domain and a PH domain (de Bruyn et al., 2000; Rebhun et al., 2000). Although RalGEF2/ RalGPS1B may not be involved in growth factor-induced RalA activation (de Bruyn et al., 2000), it probably plays an important role in other signal transduction pathways and should be monitored by a future probe. In conclusion, there is ample room for further improvement of Raichu-RalA; however, it should also be emphasized that the Raichu-RalA probe has enables us, for the first time, to visualize RalA activity and will become a powerful tool for deciphering the role of RalA in living cells. ACKNOWLEDGMENTS

Figure 8. Effect of RalA inhibition on wound closure. MDCK monolayers were wounded and, 1 h later, the cells at the wound edge were microinjected with FITC-dextran and GST, GST-RalBP1RBD, or GST-PAK-CRIB. Cells were imaged for FITC and DIC every 2 min for 4 h. (A) Overlay image of FITC and phase contrast at 4 h after microinjection and outlines the method for the quantification. The white dotted line labeled as I indicates the initial position of the wound edge. The ratio of migration distance is defined as Dn/Di, where Dn and Di are the migration distances of control cells and microinjected cells, respectively. Data from four independent experiments are averaged and shown with SD.

membrane from the endocytic cycle in the EGF-stimulated cells (Bretscher and Aguado-Velasco, 1998). Therefore, among the pleiotropic activities of RalA, the regulation of exocytosis seemingly relates most closely to the lamellipodial protrusion and also cell migration (Schmoranzer et al., 2003). Based on our observation that RalA activation is restricted to the nascent lamellipodia as well as on the previous observation that RalA activity is required for the assembly of Sec6 and Sec10 (Moskalenko et al., 2002), components of the exocyst, it could be speculated that activated RalA marks the exocytic site upon EGF stimulation. Alternatively, RalA activation at the nascent lamellipodia may regulate lamellipodial protrusion and cell migration by means of RalBP1. RalBP1 has been shown to possesses GAP activity for Rac and Cdc42 at least in vitro (Cantor et al., 1995; Jullien-Flores et al., 1995; Park and Weinberg, 1995). Thus, the effect of RalA on lamellipodial protrusion may be in part mediated by Rac and Cdc42. In addition to the effect on Rac1 and Cdc42, RalBP1 could regulate lamellipodial protrusion via two RalBP1-binding proteins, POB1 and the ␮2 medium chain of AP2 complex (Nakashima et al., 1999; Jullien-Flores et al., 2000; Oshiro et al., 2002). Both POB1 and ␮2 regulate endocytosis, and it has been reported that not only exocytosis but also endocytosis are important for the cell migration. When we used full-length RalA for the probe, the difference in FRET efficiency was ⬍5% between the wild-type and V23 mutant. The gain of the probe was remarkably in-

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We thank A. Wittinghofer, J.L. Bos, B.J. Mayer, Y. Ohta, Y. Fukui, N. Mochizuki, and A. Miyawaki for the provision of plasmids and antibodies, and N. Yoshida, N. Fujimoto, and Y. Matsuura for technical assistance. This work was supported by grants from Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Science, Sports and Culture of Japan and from the Health Science Foundation, Japan.

REFERENCES Anderson, K.E., Coadwell, J., Stephens, L.R., and Hawkins, P.T. (1998). Translocation of PDK-1 to the plasma membrane is important in allowing PDK-1 to activate protein kinase B. Curr. Biol. 8, 684 – 691. Aoki, K., Nakamura, T., and Matsuda, M. (2004). Spatio-temporal regulation of Rac1 and Cdc42 activity during nerve growth factor-induced neurite outgrowth in PC12 cells. J. Biol. Chem. 279, 713–719. Bhattacharya, M., Anborgh, P.H., Babwah, A.V., Dale, L.B., Dobransky, T., Benovic, J.L., Feldman, R.D., Verdi, J.M., Rylett, R.J., and Ferguson, S.S. (2002). beta-Arrestins regulate a Ral-GDS Ral effector pathway that mediates cytoskeletal reorganization. Nat. Cell Biol. 4, 547–555. Bhullar, R.P., and Seneviratne, H.D. (1996). Characterization of human platelet GTPase activating protein for the Ral GTP-binding protein. Biochim. Biophys. Acta 1311, 181–188. Bos, J.L. (1998). All in the family? New insights and questions regarding interconnectivity of Ras, Rap1 and Ral. EMBO J. 17, 6776 – 6782. Bretscher, M.S., and Aguado-Velasco, C. (1998). EGF induces recycling membrane to form ruffles. Curr. Biol. 8, 721–724. Cantor, S.B., Urano, T., and Feig, L.A. (1995). Identification and characterization of Ral-binding protein 1, a potential downstream target of Ral GTPases. Mol. Cell. Biol. 15, 4578 – 4584. Chardin, P., and Tavitian, A. (1986). The Ral gene: a new ras related gene isolated by the use of a synthetic probe. EMBO J. 5, 2203–2208. Chardin, P., and Tavitian, A. (1989). Coding sequences of human RalA and RalB cDNAs. Nucleic Acids Res. 17, 4380 de Bruyn, K.M., de Rooij, J., Wolthuis, R.M., Rehmann, H., Wesenbeek, J., Cool, R.H., Wittinghofer, A.H., and Bos, J.L. (2000). RalGEF2, a pleckstrin homology domain containing guanine nucleotide exchange factor for Ral. J. Biol. Chem. 275, 29761–29766. Emkey, R., Freedman, S., and Feig, L.A. (1991). Characterization of a GTPaseactivating protein for the Ras-related Ral protein. J. Biol. Chem. 266, 9703–9706. Feig, L.A., Urano, T., and Cantor, S. (1996). Evidence for a Ras/Ral signaling cascade. Trends Biochem. Sci. 21, 438 – 441. Filippa, N., Sable, C.L., Hemmings, B.A., and Van Obberghen, E. (2000). Effect of phosphoinositide-dependent kinase 1 on protein kinase B translocation and its subsequent activation. Mol. Cell. Biol. 20, 5712–5721. Fradkov, A.F., Chen, Y., Ding, L., Barsova, E.V., Matz, M.V., and Lukyanov, S.A. (2000). Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence. FEBS Lett. 479, 127–130.


RalA Activation at Nascent Lamellipodia Gildea, J.J., Harding, M.A., Seraj, M.J., Gulding, K.M., and Theodorescu, D. (2002). The role of Ral A in epidermal growth factor receptor-regulated cell motility. Cancer Res. 62, 982–985. Hamad, N.M., Elconin, J.H., Karnoub, A.E., Bai, W., Rich, J.N., Abraham, R.T., Der, C.J., and Counter, C.M. (2002). Distinct requirements for Ras oncogenesis in human versus mouse cells. Genes Dev. 16, 2045–2057. Hofer, F., Berdeaux, R., and Martin, G.S. (1998). Ras-independent activation of Ral by a Ca(2⫹)-dependent pathway. Curr. Biol. 8, 839 – 842. Itoh, R.E., Kurokawa, K., Ohba, Y., Yoshizaki, H., Mochizuki, N., and Matsuda, M. (2002). Activation of rac and cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells. Mol. Cell. Biol. 22, 6582– 6591. Jiang, H., Luo, J.Q., Urano, T., Frankel, P., Lu, Z., Foster, D.A., and Feig, L.A. (1995). Involvement of Ral GTPase in v-Src-induced phospholipase D activation. Nature 378, 409 – 412. Jullien-Flores, V., Dorseuil, O., Romero, F., Letourneur, F., Saragosti, S., Berger, R., Tavitian, A., Gacon, G., and Camonis, J.H. (1995). Bridging Ral GTPase to Rho pathways. RLIP76, a Ral effector with CDC42/Rac GTPaseactivating protein activity. J. Biol. Chem. 270, 22473–22477. Jullien-Flores, V., Mahe, Y., Mirey, G., Leprince, C., Meunier-Bisceuil, B., Sorkin, A., and Camonis, J.H. (2000). RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: involvement of the Ral pathway in receptor endocytosis. J. Cell Sci. 113, 2837–2844. Kurokawa, K., Itoh, R.E., Yoshizaki, H., Ohba, Y., Nakamura, T., and Matsuda, M. (2004). Co-activation of Rac1 and Cdc42 at lamellipodia and membrane ruffles induced by epidermal growth factor. Mol. Biol. Cell 15, 1003– 1010. Matsuda, M., Hashimoto, Y., Muroya, K., Hasegawa, H., Kurata, T., Tanaka, S., Nakamura, S., and Hattori, S. (1994). CRK protein binds to two guanine nucleotide-releasing proteins for the Ras family and modulates nerve growth factor-induced activation of Ras in PC12 cells. Mol. Cell. Biol. 14, 5495–5500. Miyawaki, A., Llopis, J., Heim, R., McCaffery, J.M., Adams, J.A., Ikura, M., and Tsien, R.Y. (1997). Fluorescent indicators for Ca2⫹ based on green fluorescent proteins and calmodulin. Nature 388, 882– 887. Miyawaki, A., and Tsien, R.Y. (2000). Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 327, 472–500. Mochizuki, N., Ohba, Y., Kobayashi, S., Otsuka, N., Graybiel, A.M., Tanaka, S., and Matsuda, M. (2000). Crk activation of JNK via C3G and R-Ras. J. Biol. Chem. 275, 12667–12671. Mochizuki, N., Yamashita, S., Kurokawa, K., Ohba, Y., Nagai, T., Miyawaki, A., and Matsuda, M. (2001). Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature 411, 1065–1068. Moskalenko, S., Henry, D.O., Rosse, C., Mirey, G., Camonis, J.H., and White, M.A. (2002). The exocyst is a Ral effector complex. Nat. Cell Biol. 4, 66 –72. Moskalenko, S., Tong, C., Rosse, C., Camonis, J., and White, M.A. (2003). Ral GTPases regulate exocyst assembly through dual subunit interactions. J. Biol. Chem., 278, 51743–51748. Nakashima, S., Morinaka, K., Koyama, S., Ikeda, M., Kishida, M., Okawa, K., Iwamatsu, A., Kishida, S., and Kikuchi, A. (1999). Small G protein Ral and its downstream molecules regulate endocytosis of EGF and insulin receptors. EMBO J. 18, 3629 –3642. Neuhaus, P., Oustanina, S., Loch, T., Kruger, M., Bober, E., Dono, R., Zeller, R., Braun, T. (2003). Reduced mobility of fibroblast growth factor (FGF)deficient myoblasts might contribute to dystrophic changes in the musculature of FGF2/FGF6/mdx triple-mutant mice. Mol. Cell. Biol. 23, 6037– 6048. Niwa, H., Yamamura, K., and Miyazaki, J. (1991). Efficient selection for highexpression transfectants with a novel eukaryotic vector. Gene 108, 193–199. Ohba, Y., Kurokawa, K., and Matsuda, M. (2003). Mechanism of the spatiotemporal regulation of Ras and Rap1. EMBO J. 22, 859 – 869. Ohba, Y., Mochizuki, N., Matsuo, K., Yamashita, S., Nakaya, M., Hashimoto, Y., Hamaguchi, M., Kurata, T., Nagashima, K., and Matsuda, M. (2000). Rap2 as a slowly responding molecular switch in the Rap1 signaling cascade. Mol. Cell. Biol. 20, 6074 – 6083. Oshiro, T., Koyama, S., Sugiyama, S., Kondo, A., Onodera, Y., Asahara, T., Sabe, H., and Kikuchi, A. (2002). Interaction of POB1, a downstream molecule

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of small G protein Ral, with PAG2, a paxillin-binding protein, is involved in cell migration. J. Biol. Chem. 277, 38618 –38626. Park, S.H., and Weinberg, R.A. (1995). A putative effector of Ral has homology to Rho/Rac GTPase activating proteins. Oncogene 11, 2349 –2355. Quilliam, L.A., Rebhun, J.F., and Castro, A.F. (2002). A growing family of guanine nucleotide exchange factors is responsible for activation of Rasfamily GTPases. Prog. Nucleic Acid Res. Mol. Biol. 71, 391– 444. Rebhun, J.F., Chen, H., and Quilliam, L.A. (2000). Identification and characterization of a new family of guanine nucleotide exchange factors for the ras-related GTPase Ral. J. Biol. Chem. 275, 13406 –13410. Rusanescu, G., Gotoh, T., Tian, X., and Feig, L.A. (2001). Regulation of Ras signaling specificity by protein kinase C. Mol. Cell. Biol. 21, 2650 –2658. Schmoranzer, J., Kreitzer, G., and Simon, S.M. (2003). Migrating fibroblasts perform polarized, microtubule-dependent exocytosis towards the leading edge. J. Cell Sci. 116, 4513– 4519. Shinohara, M., et al. (2002). SWAP-70 is a guanine-nucleotide-exchange factor that mediates signalling of membrane ruffling. Nature 416, 759 –763. Sugihara, K., Asano, S., Tanaka, K., Iwamatsu, A., Okawa, K., and Ohta, Y. (2002). The exocyst complex binds the small GTPase RalA to mediate filopodia formation. Nat. Cell Biol. 4, 73–78. Suzuki, J., Yamazaki, Y., Li, G., Kaziro, Y., and Koide, H. (2000). Involvement of Ras and Ral in chemotactic migration of skeletal myoblasts. Mol. Cell. Biol. 20, 4658 – 4665. Takai, Y., Sasaki, T., and Matozaki, T. (2001). Small GTP-binding proteins. Physiol. Rev. 81, 153–208. Tian, X., Rusanescu, G., Hou, W., Schaffhausen, B., and Feig, L.A. (2002). PDK1 mediates growth factor-induced Ral-GEF activation by a kinase-independent mechanism. EMBO J. 21, 1327–1338. Tsien, R.Y., and Miyawaki, A. (1998). Seeing the machinery of live cells. Science 280, 1954 –1955. Urano, T., Emkey, R., and Feig, L.A. (1996). Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation. EMBO J. 15, 810 – 816. van Roessel, P., and Brand, A.H. (2002). Imaging into the future: visualizing gene expression and protein interactions with fluorescent proteins. Nat. Cell Biol. 4, E15–E20. Wang, K.L., Khan, M.T., and Roufogalis, B.D. (1997). Identification and characterization of a calmodulin-binding domain in Ral-A, a Ras-related GTPbinding protein purified from human erythrocyte membrane. J. Biol. Chem. 272, 16002–16009. Wang, K.L., and Roufogalis, B.D. (1999). Ca2⫹/calmodulin stimulates GTP binding to the ras-related protein Ral-A. J. Biol. Chem. 274, 14525–14528. Ward, Y., Wang, W., Woodhouse, E., Linnoila, I., Liotta, L., and Kelly, K. (2001). Signal pathways which promote invasion and metastasis: critical and distinct contributions of extracellular signal-regulated kinase and Ral-specific guanine exchange factor pathways. Mol. Cell. Biol. 21, 5958 –5969. Wolthuis, R.M., and Bos, J.L. (1999). Ras caught in another affair: the exchange factors for Ral. Curr. Opin. Genet. Dev. 9, 112–117. Wolthuis, R.M., Franke, B., van Triest, M., Bauer, B., Cool, R.H., Camonis, J.H., Akkerman, J.W., and Bos, J.L. (1998a). Activation of the small GTPase Ral in platelets. Mol. Cell. Biol. 18, 2486 –2491. Wolthuis, R.M., Zwartkruis, F., Moen, T.C., and Bos, J.L. (1998b). Ras-dependent activation of the small GTPase Ral. Curr. Biol. 8, 471– 474. Xu, L., Frankel, P., Jackson, D., Rotunda, T., Boshans, R.L., D’Souza-Schorey, C., and Foster, D.A. (2003). Elevated phospholipase D activity in H-Ras- but not K-Ras-transformed cells by the synergistic action of RalA and ARF6. Mol. Cell Biol. 23, 645– 654. Yan, J., Roy, S., Apolloni, A., Lane, A., and Hancock, J.F. (1998). Ras isoforms vary in their ability to activate Raf-1 and phosphoinositide 3-kinase. J. Biol. Chem. 273, 24052–24056. Yoshizaki, H., Ohba, Y., Kurokawa, K., Itoh, R.E., Nakamura, T., Mochizuki, N., Nagashima, K., and Matsuda, M. (2003). Activity of Rho-family GTPases during cell division as visualized with FRET-based probes. J. Cell Biol. 162, 223–232.

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