Effect of Ral-GEF activity on neurite outgrowth of PC12 cells. PC12 cells were transiently transfected with expression plasmids under various conditions, and 3 days later the cells expressing the appropriate protein were identified by immunofluorescence. (A) Cells transfected with Ras12V37G (left) or Ras12V35S (right) were identified with α-Ras antibodies and FITC-coupled secondary antibodies. Endogenous Ras could not be detected with this antibody. All cells shown expressed exogenous Ras. (B) Cells were either untreated (left) or treated with NGF (50 ng/ml) 1 day after transfection (right), and all cells in the population were stained 2 days later with TRITC-phalloidin to detect filamentous actin. (C) Cells were transfected with Ras37G and treated 24 h later with NGF (50 ng/ml). Two days later transfected cells in the population were detected with rabbit α-Ras antibodies plus FITC-labeled secondary antibodies (left). Cells were also stained for actin with TRITC-phalloidin (right). Again, all cells shown expressed exogenous protein. (D) Cells were transfected with Ras12V37G plus MYC-Ral28N and treated with NGF as for panel C. Cells expressing Ral28N were detected with α-MYC antibodies and FITC-labeled secondary antibody (left). Cells also expressing Ras12V37G were detected with α-Ras antibodies and TRITC-labeled secondary antibodies (right panel). (E) Cells were transfected with MYC-Rgr and treated with NGF as for panel C. Transfected cells were detected with α-MYC antibodies and FITC-labeled secondary antibodies (left panel). Cells in the population were also stained for actin (right panel) with TRITC-phalloidin. (F) Cells were treated with NGF every day for 4 days and stained for actin (left panel). In a separate transfection, cells were transfected with MYC-Rgr and treated with NGF (50 ng) every day for 4 days. Transfected cells were detected with α-MYC antibodies. (G) Cells were transfected with Ras12V35S plus MYC-Rgr and treated with NGF as for panel C. Cells were stained for Ras and Rgr as described above. The label at the top left of each panel indicates the transfected gene, while the label at the bottom right of each panel indicates the protein being detected by immunofluorescence microscopy. At least 50 transfected cells were inspected for each experiment.

Effect of Ral-GEF activity on cell cycle progression of PC12 cells. (A) PC12 cells were transiently transfected with an empty expression vector or vectors encoding various signaling molecules. Twenty-four hours later, the cells were treated daily with NGF or buffer for 4 days. The cells were then exposed to 30 μg of BrdU/ml for 2 h. To identify transfected cells, a vector expressing β-galactosidase was included in all transfections. The cells were then fixed and stained with rabbit β-galactosidase antibodies to detect transfected cells and mouse anti-BrdU monoclonal antibodies to detect cells that had passed through the S phase of the cell cycle. The data are expressed as the proportion of transfected cells incorporating detectable amounts of BrdU; a representative sample of three experiments, each performed in triplicate, is shown (± standard deviations). (B) PC12 cells were grown in serum-containing medium and transiently transfected with empty vector (□) or Ral28N (●) and processed as described for panel A. The cells were treated with NGF (50 ng/ml) each day after transfection and processed at daily intervals afterward. The data represent the average of duplicate determinations. (C) Experiment performed as for panel B except that cells were grown in low-serum (0.5%) medium once NGF was added. □, Empty vector, low serum only; ▴, empty vector, low serum plus NGF; ●, Ral28N, low serum plus NGF.

Influence of Ral-GEF activity on fos gene regulation in PC12 cells. Cells were transfected with a Fos-luciferase reporter construct along with a minimal thymidine kinase promoter-Renilla reporter construct (as an internal control for transfection efficiency) and various expression plasmids. In some instances, NGF (50 ng/ml) was added to the cells 24 h later. After 2 days, firefly luciferase and Renilla luciferase activities were measured in cell lysates. The data are reported as the ratio of firefly-luciferase to Renilla luciferase activities and represents the average (± standard deviation) of at least two experiments, each performed in duplicate.

Ras effector pathways in NGF stimulated PC12 cells. Constitutive activation of Raf or PI3 kinase promotes cell cycle arrest and differentiation of PC12 cells into a neuronal phenotype. In contrast, constitutive activation of Ral-GEF promotes proliferation and blocks differentiation upon NGF stimulation. Of the two signaling molecules thought to be influenced by Ral-GEF, PLD and RalBP1, the latter appears to play a more important role in influencing neurite outgrowth in PC12 cells. NGF activates Ras and all three known Ras effector pathways. Ras and the Raf pathway are activated for hours, and this chronic activation is required for NGF-induced neurite outgrowth. In contrast, the Ral-GEF pathway (as assessed by the content of GTP-Ral) is activated by NGF for only ∼20 min. Thus, Ral-GEF signaling may function to delay the onset of cell cycle arrest and neurite outgrowth upon NGF stimulation. Dissociation of the Ral-GEF pathway from Ras activation may be necessary to allow NGF-induced differentiation of PC12 cells to occur.

Effect of Ral-GEF inhibition on neurite outgrowth of PC12 cells. PC12 cells were transiently transfected as described in the legend to Fig. 1. (A) Cells were transfected with Ral28N and treated 24 h later with NGF (50 ng/ml). Two days later the transfected cells were detected with α-Ral antibodies (left). All cells in the population were stained for F-actin with TRITC-phalloidin (right). (B) Cells were transfected with Ral28NSAAX and treated as described for panel A. (C) Cells were cotransfected with Ral28N and MYC-Ral-GDS. Twenty-four hours later the cells were treated with NGF. Two days later the cells were stained with both rabbit α-Ral antibodies and mouse α-MYC antibodies. Ral28N expression was detected with FITC-labeled anti-rabbit secondary antibodies (left), and MYC-Ral-GDS was detected with TRITC-labeled anti-mouse secondary antibodies (right).

Involvement of Rho family proteins in Ral signaling in PC12 cells. PC12 cells were transiently transfected with various expression constructs, and 24 h later the cells were treated with NGF. The cells were then processed as described in the legend to Fig. 1. (A) The cells were transfected with ΔN11Ral, and transfected cells were detected with α-Ral antibodies (left), or cells were transfected with Ral28N, and 3 days later the cells were treated with lysophosphatidic acid (2.4 μg/ml) for 90 min (right). (B) Cells were transfected with 61LCDC42 (left) or 61LRac1 (right). Three days later, the cells expressing GTPases were detected with either rabbit α-CDC42 or α-Rac1 antibody. (C) Cells were transfected with MYC-Rgr and activated 61LCDC42. Three days later, the cells were fixed and labeled with both rabbit α-CDC42 antibodies and mouse α-MYC monoclonal antibodies. CDC42 was detected with FITC-labeled anti-rabbit secondary antibodies (left), and Rgr was detected with TRITC-labeled anti-mouse secondary antibodies (right). (D) Cells were transfected with Rgr and 61LRac1 and processed as for panel B except that 61LRac-expressing cells were detected with rabbit α-Rac antibodies. (E) Cells were transfected with Ral28N plus MYC-17NCDC42. Three days later transfected cells were stained with both rabbit α-Ral antibodies and mouse anti-MYC antibodies. Ral28N expression was detected with FITC-labeled anti-rabbit secondary antibodies (left), and Rgr was detected with TRITC-labeled anti-mouse secondary antibodies (right). (F) Cells were transfected with Ral28N plus MYC-17NRac1 and processed as for panel E.

Effect of NGF on Ral-GTP levels in PC12 cells. PC12 cells or PC12-Ras17N cells were treated with NGF (50 ng/ml) for various times. The cells were lysed, and half of the lysates were incubated with glutathione agarose bound to the Ral-binding domain of RalBP1 expressed as a fusion protein with GST. The beads were washed and immunoblotted with anti-RalA or -RalB polyclonal antibodies. The other half of the cell lysates were incubated with glutathione beads bound to the Ras-binding domain of Raf expressed as a fusion protein with GST. The beads were washed and immunoblotted with anti-Ras antibody. Cell lysates from each time point were also directly immunoblotted with either anti-Ral or anti-Ras antibody to document that equal amounts of total GTPase were present in each sample. The data are representative of at least three experiments.