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.

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).

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.

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.

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.

The C₂ domain-dependent, Ca²⁺-induced plasma membrane translocation of RASAL leads to a stimulation in its ability to function as a RasGAP. (A) In vitro full-length recombinant RASAL does not function as a RasGAP. Assays were performed through the analysis of liberated [³²P]P_i as described in Materials and methods. GAP1^IP4BP was used as a positive control. Data are the means±s.e.m. from three individual determinations using H-Ras as substrate. (B) ATP stimulation activates the ability of RASAL to function as a RasGAP. Under serum-starved conditions, a significant quantity of expressed H-Ras is GTP-bound in HeLa cells transiently transfected with H-Ras. After 60 s of stimulation with 50 μM ATP, the quantity of H-Ras-GTP increases approximately two-fold. In cells expressing H-Ras and wild-type RASAL, there is a detectable decrease in the quantity of H-Ras-GTP after 60 s of stimulation with ATP (cf. RASAL−ATP with RASAL+ATP). The ability of RASAL to function as a RasGAP requires the ability to undergo Ca²⁺-induced association with the plasma membrane and a functional GAP-related domain (cf. RASAL+ATP with RASAL(D202A)+ATP and RASAL(Q507N)+ATP). (C) Quantification of H-Ras-GTP levels from HeLa cells before and 60 s after stimulation with 50 μM ATP expressed as a percentage of the pull-down in control, serum-starved cells prior to ATP stimulation (average of three separate experiments±s.e.m.).

The GFP c-Raf-1 Ras-binding domain fusion protein can be used to demonstrate RASAL-dependent Ras inactivation in live cells. (A) In HeLa cells grown in full serum, a significant amount of the GFP-RBD fluorescent reporter accumulates at the plasma membrane in cells cotransfected with the wild type (a). This is not observed in transfected cells serum starved for 12 h (b). Similar results were obtained in a further 30 cells imaged. (B) Agonist-induced dissociation of GFP-RBD from the plasma membrane. HeLa cells transfected with H-Ras, GFP-RBD, and RASAL were placed in Ca²⁺-containing extracellular medium and imaged using confocal microscopy. ATP (50 μM) was used to elevate [Ca²⁺]_i. The two images (a and b) of the cells were taken at the time points indicated on the corresponding trace. Similar results were obtained in a further five images of the cells. (C) HeLa cells transfected with H-Ras, GFP-RBD, and either RASAL(D202A) (a and b) or RASAL(Q507N) (c and d) were placed in Ca²⁺-containing extracellular media and imaged using confocal microscopy prior to (images a and c) or 30 s after (images b and d) the addition of 50 μM ATP. In each case, similar results were obtained in a further 10 images of the cells.

siRNA suppression of endogenous RASAL leads to an enhanced receptor-mediated activation of Ras. (A) HeLa cells were transiently transfected with either RASAL-specific siRNA or random siRNA. After 48 h, cells were stimulated for 2 min with 50 μM ATP, and the amount of active Ras-GTP quantified as described above. Data are expressed as a percent of control cells (average of three separate experiments±s.e.m.). (B) By RT–PCR, HeLa cells express endogenous RASAL, which, unlike GAPDH, can be suppressed following a 48 h treatment with a RASAL-specific siRNA.