c-N-Ras downregulates JNK activation and activity following treatment with apoptotic agonists. (A) N-Ras knockout fibroblasts, control N^+/+ cells, and c-N-Ras reconstituted cells were left untreated (t = 0) or treated for various times with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At the indicated times, the cells were harvested and lysates were prepared as described in the text. A 50-μg portion of protein was loaded in each lane of an SDS-10% polyacrylamide gel. Following electrophoresis and transfer, the blot was developed with anti-phospho-JNK (pJNK) and anti-mouse secondary antibody-HRP. Detection was performed using standard ECL techniques. The results are representative of three different experiments. (B) N-Ras knockout, control N^+/+, and c-N-Ras reconstituted cells were untreated or treated with TNF-α and cycloheximide as described for panel A. A 50-μg portion of protein was loaded in each lane of an SDS-10% polyacrylamide gel. Following electrophoresis and transfer, the blot was developed with anti-JNK1 rabbit polyclonal antibody and anti-rabbit secondary antibody-HRP. Detection was performed using standard ECL techniques. The results are representative of two different experiments. (C) N-Ras knockout cells, control N^+/+ cells and c-N-Ras reconstituted cells [cell lines N^−/−(2)/wtN3 and N^−/−(2)/wtN8] were left untreated or treated with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide per ml. At the indicated times, the cells were harvested and lysates prepared as described in the text. JNK1 was immunoprecipitated from 50 μg of each lysate. The immunocomplexes were incubated for 20 min at 37°C in kinase buffer. The supernatants, which contain the ATF2 substrate, were loaded onto SDS-12% polyacrylamide gels, electrophoresed, and transferred to PVDF. Signals were detected using a PhosphorImager. A nonimmune (NI) control was performed using an equal amount (1.5 μg) of purified rabbit (Rb) IgG and the 1-h TNF-α-plus-cycloheximide-treated N^+/+(1) cell lysate. The results are representative of three separate experiments. (D) N-Ras knockout cells, control N^+/+ cells, and c-N-Ras reconstituted cells [N^−/−(2)wtN3] were left untreated or treated with 1 μg of activating anti-Fas antibody per ml in the presence of 0.5 μg of protein G per ml. At the indicated times, the cells were harvested and lysates were prepared as described in the text. A 50-μg portion of each lysate was electrophoresed on an SDS-10% polyacrylamide gel and transferred to PVDF. The blot was developed with anti-pJNK monoclonal antibody and goat anti-mouse IgG-HRP as described for panel A. Detection was performed using standard ECL techniques. The positive control was N-Ras knockout cells treated for 1 h with 20 μg of anisomycin (Aniso) per ml. The results are representative of six separate experiments. (E) N-Ras knockout and control N^+/+ cells and c-N-Ras reconstituted cells were left untreated or treated with anti-Fas antibody and protein G as described for panel D. At the indicated times, the cells were harvested and lysates were prepared for immunoprecipitation of JNK as described for panel C. The immunoprecipitated JNK from each sample was used in a kinase reaction as described for panel C, using GST-ATF2 as the substrate. Following electrophoresis and transfer to PVDF, the radioactivity incorporated into the GST-ATF2 substrate was detected using a PhosphorImager. The nonimmune control (NI) used rabbit IgG and the N^+/+(1) cells treated for 1 h, as described for panel C. The results are representative of two separate experiments. (F) N-Ras knockout and control cells and N-Ras knockout cells ectopically overexpressing c-K(A)-Ras (clones 4 and 8) were left untreated or treated for 1 or 4 h with TNF-α plus cycloheximide as described for panel A. A 50-μg portion of protein was loaded in each lane of an SDS-10% polyacrylamide gel. Following electrophoresis and transfer, the blot was developed with anti-phospho-JNK (pJNK) and anti-mouse-HRP secondary antibody. Detection was performed using standard ECL techniques.

c-N-Ras downregulates the upstream kinase activators of JNK and p38. (A) (Upper panel) N-Ras knockout cells, control N^+/+ cells, and c-N-Ras reconstituted cells [N^−/− (2)/wtN3] were left untreated or treated with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At the indicated times, cell lysates were prepared and 100 μg of protein was loaded in each lane of an SDS-10% polyacrylamide gel. Following electrophoresis and transfer to PVDF, the blot was incubated with anti-phospho-MKK4 (pMKK4) polyclonal antibody. The blot was developed by using anti-rabbit secondary antibody-HRP and standard ECL techniques. The positive control consisted of N-Ras knockout cells treated for 1 h with 20 μg of anisomycin (Aniso) per ml. The results are representative of four separate experiments. (Lower panel) A 100-μg portion of the same lysates as in the upper panel was electrophoresed, transferred, and blotted with anti-MKK4 polyclonal antibody (1:1,000 dilution) followed by anti-rabbit secondary antibody-HRP. Detection was performed by standard ECL techniques. The results are representative of two separate experiments. (B) (Upper panel) N-Ras knockout, control N^+/+, and c-N-Ras reconstituted cells were left untreated or treated with TNF-α and cycloheximide as in panel A. At the indicated times, cell lysates were prepared and 100 μg of protein of each lysate was electrophoresed and transferred to PVDF. The blot was developed with anti-phospo-MKK3/6 (pMKK3/6) antibody and anti-rabbit secondary antibody-HRP. Bands were visualized by standard ECL techniques. This experiment is representative of three separate experiments. (Lower panel) Cells were left untreated or treated with TNF-α and cycloheximide as in panel A. Cell lysates (100 μg) were prepared for analysis of total MKK3 levels by using anti-MKK3 polyclonal antibody. The blot was developed by using anti-rabbit secondary antibody-HRP and standard ECL techniques. (C) N-Ras knockout and control N^+/+ cells and c-N-Ras reconstituted cells were left untreated or treated with TNF-α and cycloheximide as described for panel A. Cell lysates were prepared at the indicated times, and protein concentrations determined. A 2-μg portion of unactive His₆-tagged JNK1α1 was incubated with 12 μg of each lysate in the presence of 50 μM unlabeled ATP for 20 min at 30°C. The added His₆-JNK1α1 was isolated by incubation with nickel resin (ProBond; Invitrogen) for 45 min at 4°C. The resin was washed with p21 buffer and used in a second kinase assay to measure the activity of the isolated, recombinant JNK1α1 by using GST-ATF2 as the substrate as described in Materials and Methods. The unactive JNK1α1 incubated in the absence of lysate had no activity. The results are representative of three separate experiments. (D) Cells were left untreated or treated with TNF-α and cycloheximide as in panel A, and at the indicated times cell lysates were prepared. A 1-μg portion of unactive GST-p38α was incubated for 20 min at 30°C, as described in the text, with 12 μg of each lysate in p21 buffer containing 50 μM ATP. The GST-p38α was isolated with glutathione-agarose, and its activity was measured in a second kinase reaction using [γ-³²P]ATP with GST-ATF2 as the substrate, as described in Materials and Methods. N-Ras knockout cells were treated for 1 h with 20 μg of anisomycin per ml, and the lysate was used to activate the GST-p38α as a positive control. The unactive GST-p38α had no activity when incubated in the absence of cell lysate. (E) N-Ras knockout, control N^+/+, and c-N-Ras reconstituted cells were passaged into complete medium and 48 h later were either left untreated (t = 0) or challenged with 10 ng of EGF per ml for the times indicated. Cell lysates prepared at the indicated times were electrophoresed and transferred to PVDF. The membranes were immunoblotted for phospho-JNK (upper panel, 50 μg/lane) and pErk (Santa Cruz Biotechnology; lower panel, 20 μg/lane). The results are representative of three separate experiments.

Expression of switch 1 effector domain mutant N-Ras constructs in the N-Ras knockout cells and NIH 3T3 cells. (A) N-Ras knockout cells were transfected with three different switch 1 mutant N-Ras constructs (K61:35S, K61:37G, and K61:40C) and stable clones were selected in G418 as described in Materials and Methods. A 100-μg portion of each lysate (including the parental N-Ras knockout cells and control N^+/+ cells) was electrophoresed on SDS-13% polyacrylamide gels and transferred to PVDF. The blot was developed with anti-N-Ras monoclonal antibody and anti-mouse secondary antibody-HRP. Detection was performed by standard ECL techniques. (B) N-Ras knockout and control N^+/+ cells and N-Ras knockout cells stably expressing the switch 1 mutant N-Ras proteins were subcultured in serum-containing medium and left untreated. At 48 h after passage, the cells were harvested and lysates were prepared as described in Materials and Methods. A 20-μg portion of each lysate was electrophoresed on two separate 13% polyacrylamide gels and transferred to PVDF. The upper blot was developed with anti-phospho-Erk (pErk) and anti-mouse antibody-HRP, and the lower blot was developed with anti-Erk2 polyclonal antibody and anti-rabbit-HRP for total Erk2. Bands were visualized by standard ECL techniques. (C), NIH 3T3 cells were transfected with the indicated constructs, including the effector domain mutant N-Ras constructs, as described in Materials and Methods. (Upper panel) Expression levels of ectopic N-Ras proteins in NIH 3T3 cells was determined by Western analysis. Cultures were grown to approximately 80% confluency and then lysed in a detergent buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM NaVO₃, 1 mM dithiothreitol, 100 μM phenylmethylsulfonyl fluoride, 1 μg of aprotinin per ml, 1 μg of leupeptin per ml). Protein concentrations were determined with the bicinchoninic acid kit (Pierce). SDS-PAGE was used to resolve 40 μg of total protein from each sample on a 15% polyacrylamide gel. Proteins were transferred to Immobilon-P membranes (Millipore), and the blot was developed with anti-N-Ras monoclonal antibody and anti-mouse secondary antibody-HRP. Bands were visualized by standard ECL (Amersham) techniques. (Lower panel) Lysates prepared as described for the upper panel were used to determine the steady-state levels of Erk expression and activation. Following electrophoresis and transfer, the blot was developed with phospho-p44/42 MAP kinase (T202/Y204) E10 antibody (1:1,000; Cell Signaling Technology). Detection was performed by ECL (Amersham) following incubation with HRP-conjugated secondary anti-mouse IgG antibodies. (D) N-Ras knockout and control N^+/+ cells and N-Ras knockout cells expressing the switch 1 effector domain mutant N-Ras constructs were grown to approximately 70% confluence in complete serum-containing medium for 2 days. The cells were harvested, and lysates were prepared in the lysis buffer supplied by the manufacturer. Protein concentrations were determined as described in Materials and Methods. Immobilized Akt 1G1 monoclonal antibody (20 μl of slurry) was used to immunoprecipitate Akt from 0.5 mg of each lysate for 3 h at 4°C. The immunocomplexes were washed and used in a kinase assay as specified by the manufacturer, except that 2 μg of the substrate GSK-3 fusion protein was used per reaction. The supernatants were removed, the pellets were washed with 30 μl of buffer, and the supernatants were combined and electrophoresed on SDS-12% polyacrylamide gels. Following transfer to PVDF, the blot was developed with anti-phsopho-GSK3α/β (pGSK-3β) polyclonal antibody and goat-anti-rabbit-HRP secondary antibody followed by donkey anti-goat antibody-HRP tertiary amplification. Detection was performed using standard ECL techniques.

c-N-Ras promotes cell survival by a PI3-kinase-independent mechanism. (A) (Upper panel) N^+/+ control cells were left untreated or treated with 20 μM LY294002 for 24 or 48 h. At the indicated times, lysates were prepared as described in the text and their protein concentrations were determined. A 150-μg portion of lysate was loaded in each lane of an SDS-13% polyacrylamide gel, electrophoresed, and transferred to PVDF as described in Materials and Methods. The blot was incubated with anti-phospho-Bad (Ser¹³⁶) polyclonal antibody at a 1:250 dilution overnight at 4°C followed by anti-rabbit-HRP secondary antibody. Detection was performed using ECL-Plus. The results are representative of three separate experiments. (Lower panel) Control N^+/+(1) cells were left untreated or treated for 24 h with LY294002, and lysates were prepared. N-Ras knockout cells were left untreated and lysates were prepared as described in the text. A 100-μg portion of each lysate was loaded in each lane of an SDS-10% polyacrylamide gel, electrophoresed, and transferred to PVDF as described in the text. The blot was developed with anti-phospho-Akt (Ser⁴⁷³) primary antibody followed by anti-rabbit-HRP secondary antibody. Detection was performed by standard ECL techniques. The results are representative of two separate experiments. (B) N-Ras knockout and control N^+/+ cells in 12-well cluster plates were left untreated or pretreated for 24 h with 20 μM LY294002. Untreated or LY294002-pretreated cells were left untreated or treated for 4 h with either 1 ng of recombinant murine TNF-α per ml in the presence of 2 μg of cycloheximide (cyclo) per ml or 2 μg of cycloheximide per ml. alone. The cells were scraped in their medium and centrifuged, and the cell pellet was resuspended in 200 μl of lysis buffer provided in the Cell Death Detection ELISA Plus kit (Roche Molecular Biochemicals). The Cell Death assay was performed as described in Materials and Methods. The data are plotted as the fold increase in apoptosis compared to that of cycloheximide-treated samples. The assay was performed in triplicate and is representative of three independent experiments.

c-N-Ras downregulates p38 activation and activity following the induction of apoptosis. (A) N-Ras knockout cells, control N^+/+ cells, and c-N-Ras reconstituted cells [N^−/− (2)/wtN43A] were left untreated or treated for varying times with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At the indicated times, the cells were harvested and lysates prepared as described in the text. A 50-μg portion of each lysate was loaded onto an SDS-10% polyacrylamide gel, and following electrophoresis and transfer, the blot was developed with anti-phospho-p38 (pp38) and anti-rabbit secondary antibody-HRP. Detection was performed by standard ECL techniques. The results are representative of two separate experiments. (B) N-Ras knockout and control cells and c-N-Ras reconstituted cells were left untreated or treated with TNF-α and cycloheximide as in panel A. At the indicated times, cell lysates were prepared and 50 μg of each lysate was used to immunoprecipitate p38 as described in Materials and Methods. The resulting immunocomplexes were washed and used in a kinase assay as described in the text with 25 μCi of [γ-³²P]ATP per reaction and GST-ATF2 as the substrate. Signals were detected with a PhosphorImager. The nonimmune control (NI) consisted of immunoprecipitation of 1 h treated N^+/+(1) control cells using an equal amount of nonimmune rabbit serum. The results are representative of four separate experiments. (C) Cells were left untreated or treated with TNF-α and cycloheximide as described for panel A. At the indicated times, cell lysates were prepared and 50 μg of each lysate was used to analyze total levels of p38 using anti-p38 polyclonal antibody. The blot was developed by using anti-rabbit secondary antibody-HRP and standard ECL techniques. The results are representative of two separate experiments. (D) N-Ras knockout cells [N^−/−(3) cell line], control N^+/+ cells, and c-N-Ras reconstituted cells [N^−/−(3)/wtN5] were left untreated in complete medium or starved of serum for various times as described in Materials and Methods. At the indicated times, cell lysates were prepared. A 50-μg portion of each lysates was loaded onto an SDS-10% polyacrylamide gel, and following electrophoresis and transfer to PVDF, the blot was developed with anti-phospho-p38 and anti-rabbit secondary antibody-HRP as described in the text. Bands were visualized by standard ECL techniques. The results are representative of two separate experiments.

Artificial elevation of JNK activation increases the level of apoptosis. (A) N-Ras knockout and control N^+/+ cells stably expressing a MEKK3:ER construct and the parental cell lines were left untreated or treated for 1 h with 2 μM β-estradiol. A 50-μg portion of each cell lysate was electrophoresed, and the resultant blots were probed with anti-phospho-JNK as described in the legend to Fig. 2A. N-Ras knockout and control N^+/+ cells were treated for 1 h with 20μg of anisomycin (Aniso) per ml as a positive control. The results are representative of three separate experiments. (B) N-Ras knockout and control N^+/+ cells and their cognate clones that stably express the MEKK3:ER construct were left untreated or treated for 4 h with either 2 μM β-estradiol alone or a combination of β-estradiol and 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At 4 h of treatment, the cells were harvested, lysed, and analyzed for the level of apoptosis by a Cell Death Detection ELISA as described in Materials and Methods. Following substrate addition, readings were performed in a microtiter plate reader at λ = 405 nm. Assays were performed in triplicate, and the results are plotted with error bars representing the sample standard deviation of the mean (σn − 1). The results are representative of three separate experiments.

c-N-Ras downregulates the activation of JNK through a distinct set of downstream targets. (A) N-Ras knockout and control N^+/+ cells, c-N-Ras reconstituted cells [N^−/−(2)/wtN43A], and N-Ras knockout cells stably expressing the switch 1 mutant N-Ras constructs were left untreated or treated with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At the indicated times, cell lysates were prepared and 50 μg of each lysate was electrophoresed on SDS-10% polyacrylamide gels and transferred to PVDF. The blot was developed with anti-phospho-JNK monoclonal antibody and anti-mouse-HRP secondary antibody as described in the legend to Fig. 2A. Bands were visualized by standard ECL techniques. The results are representative of three separate experiments. (B) Cells (as described for panel A) were left untreated or treated with TNF-α and cycloheximide as described for panel A. A 50-μg portion of each lysate was electrophoresed and transferred as described previously. The blot was developed with anti-phospho-c-Jun (Ser⁶³) and anti-mouse secondary antibody-HRP. Detection was performed using standard ECL techniques. The results are representative of three separate experiments. (C) Cells were left untreated or treated with TNF-α and cycloheximide for 4 h as described for panel A. At 4 h of treatment, the cells were harvested and lysed for analysis of the level of apoptosis by a Cell Death Detection ELISA Plus as described in the legend to Fig. 1B and Materials and Methods. Assays were performed in triplicate, and the results are plotted with error bars representing the sample standard deviation of the mean (σn − 1). The results are representative of two separate experiments.