Fig. 1. (A) Status of the Ras pathway in vav3^–/– DT40 cells. Wild-type (wt) or vav3^–/– DT40 cells were stimulated with mouse anti-chicken IgM (10 µg/ml) for the indicated periods of time. Cell lysates were then obtained and analyzed to detect activated Erk1 (using anti-phospho-Erk1 immunoblots, first panel from top), Erk1 (using anti-Erk1 immunoblots, second panel from top), the GTP levels of endogenous Ras (using GST–Raf RBD pulldown experiments, third panel from top) and Ras protein (using anti-Ras immunoblots, bottom panel). WB, western blot. The fold change of activation for each protein was estimated by densitometric analysis of filters. The values (given as arbitrary units) were normalized taking into consideration the protein levels of each sample as determined by immunoblot analysis. This criterion was followed in subsequent figures. (B) Translocation of RasGRP1 to the plasma membrane in DT40 cells. A plasmid encoding an EGFP– RasGRP1 fusion protein was transfected in wild-type (upper panels) and vav3^–/– (lower panels) DT40 cells. After transfection, cells were either non-stimulated (–) or stimulated (+) as indicated above, fixed and subjected to microscopic analysis.

Fig. 3. (A) Effect of Rac1 effector mutants on RasGRP1 translocation in vav3^–/– DT40 cells. Cells were transfected with plasmids encoding EGFP–RasGRP1 and the indicated mutants of Rac1 (AU5-tagged). After transfection, cells were fixed, stained with mouse anti-AU5 antibodies and Cy3-labeled antibodies to mouse IgGs and visualized in a fluorescence microscope. Images show the distribution of the GTPases and EGFP–RasGRP1 in red and green, respectively. The areas of colocalization of those proteins are shown in yellow. (B) Activation of Ras by Rac1 effector mutants in vav3^–/– DT40 cells. Cells were transfected with a plasmid encoding HA-H-Ras either alone or in combination with vectors encoding the indicated Rac1 mutant. The levels of activation of Ras were then determined by GST–RBD pulldown experiments. The levels of expression of Rac1 and Ras were determined by immunoblotting using the indicated antibodies (right side of the panels). The mobility of each protein under analysis is indicated by an arrow on the left. The open arrow (bottom panel) signals the endogenous Rac1.

Fig. 5. (A and B) Effect of cytochalasin D in the subcellular localization of RasGRP1 in wild-type DT40 cells. Cells expressing EGFP–RasGRP1 were incubated with cytochalasin D (10 µM) for 2 h and either left non-stimulated (A, first panel) or stimulated (A and B) with anti-IgM antibodies (10 µg/ml) for 5 min. Cells were then fixed, stained with rhodamine-phalloidin (B) and subjected to microscope analysis. Images show the areas of localization of EGFP–RasGRP1 and F-actin in green (A and B) and red (B), respectively. The areas of overlap between those two proteins are shown in yellow (B). (C) Effect of cytochalasin D in the subcellular localization of RasGRP1 in COS1 cells. Cells expressing FLAG-RasGRP1 and EGFP-Vav (Δ1–186) were either left untreated (upper panels) or were treated with cytochalasin D (2 µM) for 30 min (lower panels). After this step, cells were fixed, incubated with rabbit anti-FLAG antibodies followed by Cy5-labeled antibodies to rabbit IgGs, stained with rhodamine-phalloidin and analyzed by immunofluorescence analysis. Images show the localization of EGFP-Vav (Δ1–186), FLAG-RasGRP1 and F-actin in green, blue and red, respectively. The areas of overlap between RasGRP1 and F-actin are shown in purple (right panels).

Fig. 7. (A) Effect of cytochalasin D on the activation of the Ras cascade in DT40 cells. Exponentially growing cells were cultured for 2 h in the absence (–CytD) or presence (+CytD) of cytochalasin D (10 µM) and then stimulated for 10 min with the indicated amounts of anti-IgM antibodies. Cells were then lysed and the resulting extracts analyzed by immunoblot analysis using either anti-phosphoErk1 (top panels) or anti-Erk1 (bottom panels) antibodies. The graph shows the mean and SD values of Erk1 activation obtained in three independent experiments (100% is the maximal value of activation of Erk1). (B) Effect of cytochalasin D on the Vav/Rac–RasGRP1 interaction in COS1 cells. COS1 cells expressing HA-Erk1 in the presence of the indicated proteins were incubated with cytochalasin D for 1 h before evaluating the kinase activity of Erk1 using in vitro kinase reactions (upper panel). Cell lysates from the same experiments were analyzed in parallel by anti-HA immunoblots to evaluate the expression levels of Erk1 under each experimental condition (lower panel).

Fig. 2. (A) Vav rescues the defective translocation of RasGRP1 to the plasma membrane in vav3^–/– DT40 cells. Cells were transfected with plasmids encoding Vav and EGFP–RasGRP1 and then were either left non-stimulated (–) or stimulated with anti-IgM antibodies (10 µg/ml) for 10 min. Cells were then fixed, stained with rabbit anti-Vav antibodies and Cy3-labeled antibodies to rabbit IgGs and visualized by microscopy. The two top panels show an isolated example of the majority (first panel from top) and minority (second panel from top) of cells coexpressing Vav and EGFP–RasGRP1 (see text for details). Images show the distribution of Vav and EGFP–RasGRP1 in red and green, respectively. (B) Vav rescues the defective activation of the Ras cascade in vav3^–/– DT40 cells. Cells were transfected with a plasmid encoding H-Ras (HA-tagged) either alone or in combination with a vector encoding Vav. After transfection, cells were stimulated and the levels of activation of Ras and Erk1 determined by GST–RBD pulldown experiments and anti-phosphoErk1 immunoblot, respectively. In addition, the levels of expression of each protein were determined by western blotting using the indicated antibodies. The mobility of each protein under analysis is indicated by an arrow on the left. (C) Rac1^Q61L rescues the defective translocation of RasGRP1 to the plasma membrane in vav3^–/– DT40 cells. Cells were transfected with plasmids encoding EGFP–RasGRP1 and the indicated AU5-tagged GTPases. After transfection, cells were fixed, stained with mouse anti-AU5 antibodies and Cy3-labeled antibodies to mouse IgGs and visualized in a fluorescence microscope. Images show the distribution of the GTPases and EGFP–RasGRP1 in red and green, respectively. (D) Rac1^Q61L rescues the defective activation of the Ras cascade in vav3^–/– DT40 cells. Cells were transfected with a plasmid encoding HA-tagged H-Ras either alone or in combination with vectors encoding the indicated AU5-tagged Rac1 proteins. After transfection, the levels of activation of Ras were determined by GST–RBD pulldown experiments. The levels of expression of Rac1 and Ras were determined by immunoblotting using the indicated antibodies. The mobility of each protein under analysis is indicated by an arrow on the left. The open arrow (bottom panel) signals endogenous Rac1.

Fig. 4. (A) Effect of PLC-γ inhibitors on the Ras pathway in DT40 cells. Wild-type DT40 cells were either non-stimulated (–) or stimulated (+) with anti-IgM antibodies in the absence or presence of U73343 and U73122 (10 µM each) (Alexis Biochemicals). Cells were then lysed and subjected to either GST–RBD pulldown experiments (top panel) or anti-phosphoErk1 immunoblots (third panel from top) to detect the levels of activity of Ras and Erk1, respectively. Parallel samples were analyzed by western blotting using anti-Ras (second panel from top) and anti-Erk1 (lower panel) antibodies to visualize the levels of these proteins in each condition. (B) PMA induces the translocation of RasGRP1 in DT40 cells. vav3^–/– DT40 cells expressing an EGFP–RasGRP1 fusion protein were treated for the indicated periods of time with PMA (100 nM), fixed and subjected to microscope analysis. (C) Rescue of Ras pathway activation in vav3^–/– DT40 cells by phorbol esters. Wild-type and vav3^–/– DT40 cells were stimulated for the indicated periods of time with either anti-IgM (10 µg/ml) or PMA (1 µM). The levels of activated Ras (upper panel) and phospho-Erk1 (third panel from top) were then determined as indicated in Materials and methods. Parallel samples were immunoblotted with specific antibodies to detect the levels of Ras (second panel from top) and Erk1 (bottom panel) in each condition.

Fig. 6. (A) Schematic representation of RasGRP mutants used in this study: REM, Ras exchange motif; NLS, putative nuclear localization signal; Cdc25, GDP/GTP exchange domain for Ras/Rap GTPases; EF, E–F hands; ZF, zinc finger. (B and C) RasGRP molecules associate with F-actin in cosedimentation assays. Total cell lysates from COS1 cells expressing the indicated FLAG-tagged versions of (B) RasGRP1 or (C) RasGRP2 were incubated with the indicated amounts of F-actin. Samples were then ultracentrifuged and the resulting supernatants (S) and pellets (P) were analyzed by anti-FLAG (upper panels) or anti-actin (lower panels) immunoblots after electrophoretic separation. The migration of molecular weight markers (in kDa) is shown on the left. The antibodies used in the immunoblots are indicated on the right.