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Identification of a Ras GTPase-activating protein regulated by receptor-mediated Ca2+ oscillations

2004; Springer Nature; Volume: 23; Issue: 8 Linguagem: Inglês

10.1038/sj.emboj.7600197

1460-2075

Simon Walker, Sabine Kupzig, Dalila Bouyoucef, Louise C Davies, Takashi Tsuboi, Trever G. Bivona, Gyles E. Cozier, Peter J. Lockyer, Alan Buckler, Guy A. Rutter, Maxine Allen, Mark R. Philips, Peter J. Cullen,

Cellular transport and secretion

Article1 April 2004free access Identification of a Ras GTPase-activating protein regulated by receptor-mediated Ca2+ oscillations Simon A Walker Simon A Walker Inositide Group, Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Sabine Kupzig Sabine Kupzig Inositide Group, Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Dalila Bouyoucef Dalila Bouyoucef Inositide Group, Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Louise C Davies Louise C Davies Inositide Group, Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Takashi Tsuboi Takashi Tsuboi Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Trever G Bivona Trever G Bivona Department of Medicine, Cell Biology and Pharmacology, New York University School of Medicine, New York, NY, USA Search for more papers by this author Gyles E Cozier Gyles E Cozier Inositide Group, Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Peter J Lockyer Peter J Lockyer Signalling Programme, The Babraham Institute, Babraham, Cambridge, UK Search for more papers by this author Alan Buckler Alan Buckler Ardais Corporation, One Ledgemont Center, Lexington, MA, USA Search for more papers by this author Guy A Rutter Guy A Rutter Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Maxine J Allen Maxine J Allen Oxagen Limited, Abingdon, Oxford, UK Search for more papers by this author Mark R Philips Mark R Philips Department of Medicine, Cell Biology and Pharmacology, New York University School of Medicine, New York, NY, USA Search for more papers by this author Peter J Cullen Corresponding Author Peter J Cullen Inositide Group, Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Simon A Walker Simon A Walker Inositide Group, Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Sabine Kupzig Sabine Kupzig Inositide Group, Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Dalila Bouyoucef Dalila Bouyoucef Inositide Group, Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Louise C Davies Louise C Davies Inositide Group, Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Takashi Tsuboi Takashi Tsuboi Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Trever G Bivona Trever G Bivona Department of Medicine, Cell Biology and Pharmacology, New York University School of Medicine, New York, NY, USA Search for more papers by this author Gyles E Cozier Gyles E Cozier Inositide Group, Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Peter J Lockyer Peter J Lockyer Signalling Programme, The Babraham Institute, Babraham, Cambridge, UK Search for more papers by this author Alan Buckler Alan Buckler Ardais Corporation, One Ledgemont Center, Lexington, MA, USA Search for more papers by this author Guy A Rutter Guy A Rutter Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Maxine J Allen Maxine J Allen Oxagen Limited, Abingdon, Oxford, UK Search for more papers by this author Mark R Philips Mark R Philips Department of Medicine, Cell Biology and Pharmacology, New York University School of Medicine, New York, NY, USA Search for more papers by this author Peter J Cullen Corresponding Author Peter J Cullen Inositide Group, Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Author Information Simon A Walker1, Sabine Kupzig1, Dalila Bouyoucef1, Louise C Davies1, Takashi Tsuboi2, Trever G Bivona3, Gyles E Cozier1, Peter J Lockyer4, Alan Buckler5, Guy A Rutter2, Maxine J Allen6, Mark R Philips3 and Peter J Cullen 1 1Inositide Group, Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK 2Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK 3Department of Medicine, Cell Biology and Pharmacology, New York University School of Medicine, New York, NY, USA 4Signalling Programme, The Babraham Institute, Babraham, Cambridge, UK 5Ardais Corporation, One Ledgemont Center, Lexington, MA, USA 6Oxagen Limited, Abingdon, Oxford, UK *Corresponding author. Inositide Group, Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK. Tel.: +44 117 954 6426; Fax: +44 117 928 8274; E-mail: [email protected] The EMBO Journal (2004)23:1749-1760https://doi.org/10.1038/sj.emboj.7600197 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Receptor-mediated increases in the concentration of intracellular free calcium ([Ca2+]i) are responsible for controlling a plethora of physiological processes including gene expression, secretion, contraction, proliferation, neural signalling, and learning. Increases in [Ca2+]i often occur as repetitive Ca2+ spikes or oscillations. Induced by electrical or receptor stimuli, these repetitive Ca2+ spikes increase their frequency with the amplitude of the receptor stimuli, a phenomenon that appears critical for the induction of selective cellular functions. Here we report the characterisation of RASAL, a Ras GTPase-activating protein that senses the frequency of repetitive Ca2+ spikes by undergoing synchronous oscillatory associations with the plasma membrane. Importantly, we show that only during periods of plasma membrane association does RASAL inactivate Ras signalling. Thus, RASAL senses the frequency of complex Ca2+ signals, decoding them through a regulation of the activation state of Ras. Our data provide a hitherto unrecognised link between complex Ca2+ signals and the regulation of Ras. Introduction Many receptor tyrosine kinases and G-protein-coupled receptors are linked to the generation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through the phosphoinositide-specific phospholipase C (PLC)-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). Once generated, IP3 stimulates the release of calcium (Ca2+) from internal stores thereby giving rise to an increase in the concentration of intracellular free Ca2+ ([Ca2+]i). The increase in [Ca2+]i is responsible for controlling a plethora of cellular processes such as fertilisation, secretion, contraction, proliferation, neural signalling, and learning (Bootman et al, 2001; Berridge et al, 2003). Understanding how such a simple ion can regulate so many diverse cellular processes is a key goal of Ca2+ and cell biologists. The answer seems to lie in the coupling of the Ca2+ signal, in terms of speed, amplitude, and spatiotemporal patterning, to an extensive molecular repertoire of Ca2+ sensing pathways that regulate cell physiology (Berridge et al, 2003). Receptor-mediated increases in [Ca2+]i are often observed as repetitive Ca2+ spikes or oscillations that increase their frequency with the amplitude of the receptor stimuli, a phenomenon that appears critical for the induction of selective cellular functions (Bootman et al, 2001; Berridge et al, 2003). Indeed, the frequency of Ca2+ oscillations determines the efficiency of mitochondrial ATP production (Hajnoczky et al, 1995), and gene expression driven by the transcription factors NF-AT, OAP, and NF-κB (Dolmetsch et al, 1998; Li et al, 1998). To use such 'frequency modulation', cells have developed decoders that respond to the frequency and duration of the Ca2+ signal. At present, however, the only known examples of such decoders are calmodulin (Craske et al, 1999), protein kinase C (Oancea and Meyer, 1998; Mogami et al, 2003; Violin et al, 2003) and calmodulin-dependent protein kinase II (De Koninck and Schulman, 1998; Bayer et al, 2002). Here we describe the characterisation of RASAL, a Ras GTPase-activating protein, as a decoder of complex Ca2+ signals. Ras proteins are binary molecular switches that, by cycling between inactive GDP-bound and active GTP-bound forms, regulate multiple cellular signalling pathways including those that control growth and differentiation (Bivona and Philips, 2003; Downward, 2003; Hancock, 2003; Hingorani and Tuveson, 2003). The extent and duration of Ras activation are governed by the interplay between guanine nucleotide exchange factors (GEFs), which induce the dissociation of GDP to allow association of the more abundant GTP, and GTPase-activating proteins (GAPs), which bind to the GTP-bound form and enhance the intrinsic Ras GTPase activity (Bivona and Philips, 2003; Downward, 2003; Hancock, 2003; Hingorani and Tuveson, 2003). The best-characterised upstream signals known to activate Ras emanate from tyrosine kinase-linked receptors. Stimulation of these receptors induces the docking of specific RasGEFs such as mSOS and RasGAPs like p120GAP to the activated receptor, where they coordinate the overall activation of Ras (Bivona and Philips, 2003; Downward, 2003; Hancock, 2003; Hingorani and Tuveson, 2003). However, many other receptor types, including G-protein-coupled receptors, can also activate Ras. In some cases, this has been shown to involve transactivation of growth factor receptor tyrosine kinases, and in others a role for [Ca2+]i has been proposed (reviewed in Cullen and Lockyer, 2002). Direct evidence for a role of Ca2+ in the regulation of Ras signalling has come with the identification of Ca2+-regulated RasGEFs and -GAPs (Cullen and Lockyer, 2002). Ras-GRF1 (Martegani et al, 1992; Shou et al, 1992; Farnsworth et al, 1995) and a closely related protein Ras-GRF2 (Fam et al, 1997) function as Ca2+/calmodulin-dependent RasGEFs. A distinct family of GEFs that act on Ras and the related protein Rap1, the GRP/CalDAG-GEF family (Cullen and Lockyer, 2002), is regulated not only by Ca2+ but also DAG. This is a four-gene family encoding five GEFs: Ras-GRP1/CalDAG-GEFII, CalDAG-GEFI, Ras-GRP2, Ras-GRP3/CalDAG-GEFIII, and Ras-GRP4 (Ebinu et al, 1998; Kawasaki et al, 1998; Tognon et al, 1998; Clyde-Smith et al, 2000; Ohba et al, 2000; Yamashita et al, 2000; Lorenzo et al, 2001). Some of these proteins display sensitivity to Ca2+ through direct binding of Ca2+ to a pair of carboxy-terminal atypical EF hands, that is, GRP1, CalDAG-GEFI, and GRP2 (Ebinu et al, 1998; Kawasaki et al, 1998). A great deal of evidence has also accumulated for the existence of Ca2+-stimulated RasGAPs. For example, p120GAP has been reported to be regulated by Ca2+ (Filvaroff et al, 1992; Gawler et al, 1995a,1995b), although others have questioned this conclusion (Clark et al, 1995). The prototypical Ca2+-triggered RasGAP is CAPRI (Lockyer et al, 2001), a member of the GAP1 family (Maekawa et al, 1993, 1994; Baba et al, 1995; Cullen et al, 1995; Yamamoto et al, 1995; Lockyer et al, 1997, 1999; Allen et al, 1998; Minagawa et al, 2001; Walker et al, 2002). Members of this family, which includes RASAL (Allen et al, 1998), share a common molecular architecture of N-terminal tandem C2 domains, a C-terminal pleckstrin homology domain adjacent to a Bruton's tyrosine kinase motif, and a central catalytic RasGAP-related domain. In unstimulated cells, CAPRI is a cytosolic, inactive RasGAP (Lockyer et al, 2001). However, upon agonist-evoked elevation in [Ca2+]i, CAPRI undergoes a rapid, C2 domain-dependent association with the plasma membrane (Lockyer et al, 2001). Importantly, this membrane association activates the RasGAP activity of CAPRI (Lockyer et al, 2001). In the present study, we show that like CAPRI, RASAL is a cytosolic protein that undergoes a rapid translocation to the plasma membrane in response to receptor-mediated elevation in [Ca2+]i, a translocation that activates its ability to function as a RasGAP. However, unlike CAPRI, which undergoes a transient plasma membrane association upon receptor stimulation (Lockyer et al, 2001), and does not sense oscillations in [Ca2+]i (P Lockyer, personal communication, 2003), we show that RASAL oscillates between the plasma membrane and the cytosol in synchrony with simultaneously measured repetitive Ca2+ spikes. We propose therefore that RASAL constitutes a molecular machine that can sense the frequency of complex Ca2+ oscillations decoding into a dynamic regulation in the activation of Ras. Results Tandem C2 domains of RASAL bind phosphatidylserine- and phosphatidylcholine-enriched liposomes in a Ca2+-dependent manner A feature of the RASAL tandem C2 domains is their similarity to the well-characterised, high-affinity Ca2+-dependent, phospholipid-binding C2 domains from synaptotagmin III and protein kinase C βII (Figure 1A). Indeed, each RASAL C2 domain has a fully conserved consensus sequence necessary for C2 domains to bind phospholipids in a Ca2+-dependent manner (Cho, 2001). To examine whether the C2 domains of RASAL were capable of Ca2+-dependent phospholipid binding, we investigated the ability of RASAL to associate with sucrose-loaded liposomes composed either of phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, or phosphatidylcholine. Although no binding of a recombinant RASAL glutathione-S-transferase (GST) fusion protein was detected to liposomes in the absence of Ca2+, addition of sufficient total Ca2+ to elevate the free Ca2+ content to 100 μM resulted in the association of RASAL with phosphatidylcholine- and phosphatidylserine-containing liposomes (Figure 1B). To examine the C2 domain dependency of the Ca2+-induced association, we generated a series of RASAL deletion mutants. GST-ΔC2A-RASAL, GST-ΔC2B-RASAL, and GST-ΔC2-RASAL all failed to undergo Ca2+-dependent binding to phosphatidylcholine- or phosphatidylserine-containing liposomes (Figure 1C). Consistent with both C2 domains being required for efficient Ca2+-induced association were the observations that site-directed mutagenesis of key aspartate residues either within the C2A, namely D21A, or the C2B domain, D202A, inhibited the Ca2+-dependent association of the corresponding full-length RASAL (Figure 1C). Figure 1.The tandem C2 domains of RASAL bind phosphatidylcholine-containing liposomes in a Ca2+-dependent manner. (A) The alignment of RASAL C2A (top) and (bottom) C2B domains with the corresponding domains from CAPRI, synaptotagmin III (Syt III), and protein kinase CβII (PKCβII). Predicted β strands are indicated. The arrows show the positions of aspartate side chains that form the three Ca2+-binding sites in the synaptotagmin I C2A domain. (B) Association of full-length recombinant RASAL with either phosphatidylcholine (PC)-, phosphatidylethanolamine (PE)-, phosphatidylinositol (PI)-, or phosphatidylserine (PS)-containing liposomes when assayed with a free Ca2+ concentration of either <1 nM or 100 μM. Formation of liposomes and the resolution of the resultant RASAL/lipid complex were achieved as described in Materials and methods. (C) Quantification of the Ca2+-dependent association of RASAL with PC-containing liposomes highlighting the important role of the tandem C2 domains. Binding was analysed using recombinant RASAL lacking either the individual C2A (ΔC2ARASAL) or C2B (ΔC2BRASAL) domains, both C2A and C2B domains (ΔC2RASAL), or point mutants targeting key aspartate residues in either the C2A (RASAL(D21A)) or C2B (RASAL(D202A)) domains. Similar data were obtained using PS-containing liposomes. Download figure Download PowerPoint RASAL undergoes a C2 domain-dependent association with the plasma membrane following a receptor-induced elevation in [Ca2+]i To determine the physiological significance of the ability of the RASAL tandem C2 domains to undergo Ca2+-dependent phospholipid association, we observed the subcellular localisation of RASAL prior to and during stimulation with agonists that elevated the [Ca2+]i concentration. Figure 2A shows images of a HeLa cell expressing a green fluorescent protein (GFP)-tagged RASAL chimaera prior to and during stimulation with the muscarinic agonist carbachol. The four images, taken immediately before and 1, 2, and 3 s after stimulation, clearly show a receptor-induced plasma membrane translocation of GFP-RASAL. To analyse the kinetics of the translocation process in more detail, the relative increase in plasma membrane over cytosolic fluorescence intensity (R) was calculated for a series of 300 images captured at one frame a second (Figure 2B). This relative plasma membrane translocation parameter was plotted as a function of time for each cell analysed. Figure 2.RASAL undergoes receptor-induced plasma membrane translocation. (A) Receptor activation induces the translocation of GFP-RASAL to the plasma membrane. The images shown were recorded from a HeLa cell after the addition of carbachol (100 μM). Numbers indicate time in seconds, with the initial frame arbitrarily labelled 0, prior to translocation. (B) Definition of the relative plasma membrane translocation parameter (R). (C) Release of calcium from internal stores is sufficient for the receptor-induced translocation of GFP-RASAL to the plasma membrane. Traces are taken from two individual HeLa cells stimulated with carbachol (100 μM) either in the presence (solid line) or absence (dashed line) of extracellular calcium, and are representative of responses seen in 10 of 31 and 15 of 24 cells imaged, respectively. (D) Examples of GFP-RASAL plasma membrane translocation in a variety of cell lines stimulated with different agonists in the presence of extracellular calcium. (i) CHO cells stimulated with ATP (50 μM); (ii) COS cells stimulated with histamine (100 μM); (iii) COS cells stimulated with ATP (50 μM). (E) Receptor-induced translocation requires the tandem C2 domains of RASAL. Traces are taken from individual HeLa cells transiently transfected with GFP chimaeras of the different RASAL mutants and are representative of the typical responses seen (n⩾10 in each case). Download figure Download PowerPoint For maximal receptor stimuli using 100 μM of carbachol, translocation was observed to be transient in nature (Figure 2C). Translocation required an elevation in [Ca2+]i since it could be mimicked by addition of the Ca2+ ionophore ionomycin (data not shown), and was blocked by pretreatment with the intracellular Ca2+ chelator BAPTA-AM (data not shown). In addition, when the transfected cells were stimulated in the absence of extracellular Ca2+, the plasma membrane translocation of GFP-RASAL could still be observed, although association reversed typically after a period of 30 s or less (Figure 2C). Thus, Ca2+ release from internal stores is capable of inducing the plasma membrane association of RASAL. The receptor-mediated plasma membrane association of RASAL was also observed in a variety of other cell types stimulated with a range of agonists (Figure 2D). Consistent with a requirement for the C2 domains in the Ca2+-induced association were the observations that neither ΔC2RASAL nor the D21A or D202A mutants underwent significant plasma membrane association on stimulation with 100 μM of carbachol (Figure 2E). These data clearly demonstrate that the tandem C2 domains confer upon RASAL an ability to undergo plasma membrane association following an elevation in [Ca2+]i. It should be noted that in contrast with the pleckstrin homology domain-dependent spatial regulation of GAP1IP4BP and GAP1m (Lockyer et al, 1997, 1999; Cozier et al, 2000a, 2003), RASAL failed to associate with the plasma membrane following epidermal growth factor stimulation of PC12 cells or after cotransfection of HeLa cells with a constitutively active phosphoinositide 3-kinase (data not shown). This indicates that human RASAL is unlikely to have a high affinity for phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) or phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). These data are entirely consistent with the human RASAL pleckstrin homology domain missing several of the key residues known to be required for binding to these phosphoinositides (Cozier et al, 2000a,2000b, 2003). RASAL undergoes an oscillatory translocation to the plasma membrane that occurs in synchrony with repetitive Ca2+ spikes Strikingly, in 21 out of the 31 HeLa cells imaged, stimulation with carbachol resulted in an oscillatory plasma membrane association of RASAL (Figure 3A, Supplementary data). Both sinusoidal-like and baseline oscillation were observed (Figure 3B). Furthermore, oscillatory membrane association was frequency encoding as submaximal agonist doses induced slower oscillations in membrane association (Figure 3B). Such an oscillatory plasma membrane association is reminiscent of receptor-triggered Ca2+ spikes (Bootman et al, 2001; Berridge et al, 2003). In order to investigate this phenomenon further, we measured changes in [Ca2+]i simultaneously with the subcellular localisation of RASAL. To achieve this, HeLa cells expressing GFP-RASAL were loaded with the Ca2+-sensitive indicator Fura-2, and the receptor-induced change in the fluorescence of the Ca2+ indicator (measured by excitation at 380 nm) was imaged simultaneously with the membrane association of GFP-RASAL (excitation at 488 nm). Using a wide-field imaging system, we could clearly observe the receptor-induced association of RASAL with the plasma membrane (Figure 4A). Capturing simultaneous pairs of images at 380 and 488 nm allowed oscillatory increases in [Ca2+]i to be imaged with translocation of GFP-RASAL. In Figure 4, a correlation can be observed between the Ca2+ spikes elicited by addition of 100 μM carbachol and the oscillatory association of RASAL with the plasma membrane. RASAL is therefore capable of sensing the dynamic receptor-mediated oscillations in [Ca2+]i through a synchronous oscillatory association with the plasma membrane. Figure 3.Receptor-induced translocation of RASAL occurs in an oscillatory manner. (A) Selected individual frames taken from HeLa cells showing oscillatory plasma membrane association of GFP-RASAL during stimulation with 100 μM of carbachol. Numbers indicate time in seconds, with the initial frame arbitrarily labelled 0, prior to the first translocation. A movie of this oscillatory translocation is available as Supplementary Material. (B) GFP-RASAL translocations in HeLa cells, showing (i) sinusoidal and (ii, iii) baseline oscillatory responses after addition of 100 or 1 μM of carbachol. Download figure Download PowerPoint Figure 4.Oscillations in the translocation of RASAL are synchronous with repetitive Ca2+ spikes. (A) Parallel measurements of intracellular Ca2+ and GFP-RASAL plasma membrane translocation. HeLa cells expressing GFP-RASAL were loaded with Fura-2 AM and stimulated with carbachol (100 μM) in the presence of extracellular Ca2+. Fluorescence intensities were recorded sequentially at excitation wavelengths of 380 nm for Fura-2 (a decrease in fluorescence intensity indicating an increase in intracellular Ca2+ concentration) and 488 nm for GFP-RASAL. The first frames of the two wavelengths are labelled 0 s just after the addition of agonist. (B) Time course comparing intracellular Ca2+ with plasma membrane translocation of GFP-RASAL. Images recorded were of insufficient quality to determine R values, hence GFP-RASAL translocation was measured by averaging pixel intensities from a cytoplasmic region of interest. Similarly, Fura-2 fluorescence was measured by averaging pixel intensities from a region of interest. Average pixel intensities for the two excitation wavelengths are shown as inverse changes against time, that is, the average pixel intensity for the region of interest from each frame was compared to the first imaged and the relationship plotted as an inverse against time to represent increases in intracellular Ca2+ and increases in GFP-RASAL translocation. Download figure Download PowerPoint Ca2+-induced plasma membrane translocation of RASAL occurs in the form of translocation waves Besides the temporal aspect of complex Ca2+ signalling that is manifested as repetitive Ca2+ spikes, each individual Ca2+ spike has a spatial component that is observed as an elevated wave of Ca2+ that originates from a localised region prior to spreading throughout the cell (Bootman et al, 2001; Berridge et al, 2003). To examine the spatial aspects of the receptor-induced plasma membrane association of RASAL, we employed total internal reflection fluorescence (TIRF) microscopy. In TIRF imaging, a laser beam is totally internally reflected from the glass–water interface and generates an exponentially decaying excitation field (an evanescent wave) with a penetration depth of approximately 70 nm. This method selectively excites fluorescent molecules at or near the plasma membrane that is attached to the coverslip, with only a minimal excitation of the cytosolic molecules. Translocation of GFP-RASAL from the cytosol to the plasma membrane therefore results in an increase in fluorescence intensity since the protein comes from the dark cytosol into the evanescent wave field near the plasma membrane. Compared to confocal microscopy, TIRF imaging significantly increases the signal-to-noise for plasma membrane translocation such that greater spatial and temporal information on translocation to the plasma membrane can be obtained. In HeLa cells expressing GFP-RASAL, the addition of 50 μM ATP resulted in a significant increase in fluorescence intensity at the plasma membrane, and in most of the series of TIRF images analysed (n=3 out of 4), the increase was repetitive (Figure 5A). These data are consistent with the ability of RASAL to sense the dynamic receptor-mediated oscillations in [Ca2+]i described above. More detailed analysis of a series of TIRF images revealed that the repetitive increase in GFP-RASAL fluorescence observed upon receptor stimulation occurred in the form of fluorescent waves that originated from discrete fluorescent 'hot' spots and propagated across the cell (Figure 5B). Thus the receptor-induced plasma membrane association of RASAL has both complex temporal and spatial properties that are driven by the intricate spatiotemporal patterning of intracellular Ca2+ signals. Figure 5.TIRF microscopy reveals spatial aspects of GFP-RASAL plasma membrane recruitment. (A) An evanescent wave was used to excite GFP-RASAL at the plasma membrane of a HeLa cell (within approximately 70 nm of the coverslip surface) during stimulation with 50 μM of ATP. Time course shows oscillations in GFP-RASAL plasma membrane recruitment. The inset shows the cell imaged and the region of interest from which the pixel intensities were taken for the trace. (B) An evanescent wave was used to excite GFP-RASAL at the plasma membrane of a HeLa cell. The cell was stimulated by addition of ATP (50 μM), with the first image shown taken immediately before GFP-RASAL translocation (arbitrarily labelled as 0). Subsequent frames were taken every 0.2 s. In the pseudocolour images, the association of GFP-RASAL can be seen to recruit from a 'hot' spot centre right of the cell and rapidly propagate out. Download figure Download PowerPoint Ca2+-induced plasma membrane translocation of RASAL enhances its ability to function as a RasGAP Given that RASAL contains a conserved RasGAP-related domain (Allen et al, 1998), we addressed the physiological significance of the Ca2+-induced plasma membrane association by investigating the ability of RASAL to function as a RasGAP. Using an in vitro assay, we failed to detect any RasGAP activity for recombinant RASAL (Figure 6A). Given the precedent set by the demonstration that CAPRI only functions as a RasGAP following its Ca2+-induced association with the plasma membrane (Lockyer et al, 2001), we assayed the effect of the Ca2+-dependent membrane association on the ability of RASAL to function as an in vivo RasGAP. To achieve this, we transiently transfected HeLa cells with RASAL and H-Ras and assayed the level of Ras-GTP u