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Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio,1 Department of Pharmacology,2 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina3
Received 26 June 2001/ Returned for modification 20 August 2001/ Accepted 15 November 2001
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ras-GTP transmits its signal through interactions with a large number of target proteins. These include the Raf kinases (Raf-1, B-Raf, and A-Raf) (77, 78, 118, 120, 124, 136), phosphatidylinositol 3-kinase (PI3-kinase) (96, 97), and RalGDS family members (44, 58, 109, 130). Other candidate Ras effector proteins have been identified and include AF-6, Canoe, Rin-1, Nore-1, PKC
, and phospholipase C
(PLC) (10, 18, 54, 69, 106).
Studies using recombinant proteins suggested that all the Ras isoforms are capable of binding the same effectors with similar affinities (41). Mounting evidence, however, suggests that this is not the case in whole cells (37, 69, 129, 133). One study reported that oncogenic K-Ras more potently activated Raf-1 than G12V-Ha-Ras in transfected COS cells (133). Our laboratory has demonstrated that Raf-1 preferentially binds to c-N-Ras in G12V-Ha-Ras-transformed C3H10T1/2 fibroblasts (37). We have also observed that c-N-Ras promotes cell survival through an Akt-dependent pathway whereas neither c-K(A)-Ras nor c-K(B)-Ras could substitute for this function (129).
Through interactions with downstream effectors, Ras proteins perform functional roles in a large number of biological processes including cell proliferation, transformation, cell cycle progression, migration, differentiation, immune responses, apoptosis, and cell survival (10, 26, 28, 69, 129). Because Ras is positioned as a central molecular switch in the coordinated regulation of multiple biological outcomes, it must interact with a variety of downstream targets to exert its effects through cellular signaling pathways that ultimately influence the cell fate. In fact, the use of selective effector-binding mutant Ras proteins demonstrated that Ras must interact with multiple downstream targets to generate a transformed phenotype (129).
The role of Ras and Ras-dependent signaling pathways in apoptosis and cell survival is the subject of intense investigation. Many of these studies have used ectopic expression of oncogenic Ras to examine whether Ras promotes or inhibits apoptosis. A few studies have reported that oncogenic Ras acts to induce apoptosis (12, 66, 67, 81, 83, 116). In contrast, a growing body of evidence suggests that oncogenic Ras prevents apoptosis and promotes cell survival (70, 89, 91, 93, 98, 103, 112). The actual outcome of oncogenic Ras signaling in promoting or inhibiting apoptosis may be influenced by the level of Ras expression, the specific cell system, and/or the presence or absence of survival or other factors (28).
The effects of Ras on cell survival and apoptosis may be mediated by signaling through different pathways. Signaling through Raf-1/MEK/ERK is proapoptotic in some circumstances (32, 51). However, it has also been reported that the Ras/Raf/MEK/ERK pathway also functions in preventing apoptosis (6, 31, 53, 76, 105). Recent reports suggest that additional functions of Raf-1, independent of MEK and ERK activities, are involved in counteracting the induction of apoptosis (47, 73).
A large body of evidence supports a survival function for Ras by its signaling through the PI3-kinase/Akt pathway (86, 99, 129). Akt is thought to promote cell survival, in part, through phosphorylation and inactivation of proapoptotic Bad and caspase-9 (19, 28, 56). It has been reported that in some circumstances, promotion of cell survival by Ras proceeds through both the Raf-1/MEK/ERK and PI3-kinase pathways (42, 132). A recent report also indicates that oncogenic Ras inhibits anoikis-induced cell death through stabilization of BclxL expression and that this effect was independent of either ERK or PI3-kinase (98). This suggests that, as with transformation (125), Ras may mediate cell survival through multiple pathways.
In an effort to dissect distinct Ras isoform-specific functions, we have previously demonstrated that c-N-Ras promotes cell survival through an Akt-dependent pathway. In contrast to control cells (N+/+), N-Ras knockout fibroblasts (N-/-) possess low levels of steady-state phosphorylated Bad and are highly sensitive to the induction of apoptosis. Ectopic expression of c-N-Ras in the N-Ras knockout cells restores pBad levels to those observed in control cells and rescues the apoptotically sensitive phenotype (129). Ectopic expression of either c-K(A)- or c-K(B)-Ras could not reverse the heightened apoptotic sensitivity of the parental N-Ras knockout cells. Experiments with inhibitors of PI3-kinase, which also inhibit Akt signaling, suggested that c-N-Ras performs additional, Akt-independent functions that promote cell survival.
The present study is aimed at elucidating other mechanisms by which c-N-Ras promotes cell survival. Using wild-type and selective effector-binding domain mutants of N-Ras, we have found that c-N-Ras provides protection from apoptosis by attenuation of Jun N-terminal protein kinase (JNK) activity. This survival function operates independently of signaling through either Raf-1/MEK/ERK or PI3-kinase (p110
or p110ß).
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1, and full-length inactive GST-tagged p38 were from Upstate Biotechnology, Inc. (Lake Placid, N.Y.). Phosphospecific c-Jun (Ser63) monoclonal, c-Jun monoclonal, phospho-JNK monoclonal, phospho-Erk monoclonal, N-Ras monoclonal, Erk1 polyclonal, Erk2 polyclonal, MKK4 polyclonal (MEK-4), and JNK1 polyclonal antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). Hamster anti-mouse Fas receptor antibody (clone Jo2) was from BD Pharmingen (San Diego, Calif.). Anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP) was from BD Transduction Laboratories (Lexington, Ky.). Anti-mouse antibody conjugated to HRP was from Kirkegaard & Perry Laboratories (Gaithersburg, Md.). Protein G and ß-estradiol were from Sigma-Aldrich Chemical Co (St. Louis, Mo.). Nonimmune rabbit immunoglobulin G (IgG) was from Pierce Chemical Co. (Rockford, Ill.), and nonimmune rabbit serum was from Biodesign International (Saco, Maine). Anti-p38 rabbit serum (for immunoprecipitation) was a generous gift from Andrew C. Larner (Cleveland Clinic Foundation, Lerner Research Institute). The Akt kinase assay kit was from Cell Signaling Technology. Cell culture. N-Ras knockout (N-/-) and control N+/+ mouse embryo fibroblasts were a generous gift from R. Kucherlapati (Albert Einstein College of Medicine) (117). The fibroblasts were immortalized as previously described (129). To avoid any cell-specific changes arising from immortalization, multiple independently isolated cell lines were used throughout these studies. Independently derived N-Ras knockout cell lines are designated N-/-(#), where the number indicates a different cell line. N+/+ control cell lines are also designated with numbers to indicate independently immortalized cell lines [e.g., N+/+(1)]. Cells were grown in complete medium consisting of Dulbecco's modified Eagle's medium (DMEM) containing 12% fetal bovine serum (Atlanta Biologicals, Norcross, Ga.), 1x antibiotic/antimycotic, and 1x nonessential amino acids (Life Technologies, Inc.-Invitrogen, Rockville, Md.). Cells stably expressing an estrogen-responsive MEKK3 construct (MEKK3:ER) were grown in phenol red-free DMEM (Life Technologies, Inc.-Invitrogen) containing sodium bicarbonate, 1 mM sodium pyruvate (Sigma), 1x antibiotic/antimycotic, 1x nonessential amino acids, 10% charcoal-stripped fetal bovine serum (Atlanta Biologicals), and 100 µg of G418 per ml. The cells were kept in complete medium for all experiments unless otherwise stated. Serum starvation was performed by rinsing cells with phosphate-buffered saline (PBS) (20 mM Na2HPO4, 120 mM NaCl [pH 7.4]) and incubating them in DMEM containing nonessential amino acids and antibiotic/antimycotic solution.
Pharmacological treatments.
Recombinant murine tumor necrosis factor alpha (TNF-) (Calbiochem-Novabiochem Corp., La Jolla, Calif.) was dissolved in 0.2-µm-pore-size filtered PBS containing 0.1% bovine serum albumin (Sigma-Aldrich) and stored in aliquots at -80°C. The Fas receptor was activated by incubation of cells for the indicated times in complete medium containing 1 µg of anti-Fas receptor antibody (clone Jo2, form NA/LE; BD Pharmingen) per ml and 0.5 µg of protein G (Sigma-Aldrich) per ml. LY294002 was from Calbiochem-Novabiochem and was used at a final concentration of 20 µM.
Cloning and transfections. c-N-Ras/pIBW3 containing the c-N-Ras gene under the control of the thymidine kinase promoter was a generous gift from Angel Pellicer (New York University). N-Ras knockout cells ectopically expressing c-N-Ras at control N+/+ cell levels were prepared as described previously and maintained in complete medium containing 200 µg of G418 per ml (129). N-Ras knockout cells ectopically expressing c-K(A)-Ras or c-K(B)-Ras were generated by transfection of these constructs in a pTargeT vector containing a cytomegalovirus promoter (Promega, Madison, Wis.) and maintenance of stable clones in 200 µg of G418 per ml (129).
The MEKK3:ER/pcDNA3 construct was a generous gift from Ulrich Siebenlist (National Institutes of Health). N-Ras knockout and control N+/+ cells were transfected with 200 ng of MEKK3:ER/pcDNA3 using Lipofectamine-Plus (Invitrogen/Life Technologies), and stable clones were selected in 450 µg of G418 per ml. Positive clones were detected by immunoblotting with an anti-estrogen receptor (ER) antibody (data not shown). Stable clones were maintained in phenol red-free DMEM as described above.
(i) Generation of N-Ras(61K) effector domain mutant constructs. We introduced missense mutations into the appropriate regions of cDNA to generate the 35S, 37G, and 40C N-Ras effector domain mutants. cDNA sequences encoding the effector domain mutants were generated with the QuikChange site-directed mutagenesis kit (Stratagene) and the cDNA sequence for N-Ras(61K) in the pBluescript II KS(+)-N-ras(61K) plasmid as a template for mutagenesis. The mutations at position 35, 37, and 40 were created by changing a threonine to serine, a glutamic acid to glycine, and a tyrosine to cysteine at these amino acid locations, respectively. The pBluescript II KS(+)-N-ras(61K) template was constructed by inserting the cDNA encoding N-Ras(61K) into the BamHI site of pBluescript II KS(+) (Stratagene). The N-ras(61K) cDNA was obtained by digesting the previously generated and characterized pCMVneo-N-ras(61K) with BamHI (92). After mutagenesis, all constructs were confirmed by sequencing. The BamHI fragment from each effector domain mutant was inserted into the BamHI site of the pZIP-NeoSV(x)1 retrovirus mammalian expression vector (11a). The orientation was verified by sequencing.
(ii) Establishment of N-Ras knockout fibroblasts expressing effector domain mutants in N-Ras knockout cells. N-Ras knockout cells were transfected as described above with 300 ng of each of the N-Ras effector domain mutant constructs, and stable clones were selected in 450 µg of G418 per ml. Individual clones were maintained in DMEM containing 12% fetal bovine serum, 1x antibiotic/antimycotic, and 200 µg of G418 per ml. Levels of expression were confirmed by Western analysis (see Fig. 6).
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Preparation of cell lysates. All lysis buffers contained the phosphatase inhibitors 30 mM ß-glycerophosphate, 5 mM p-nitrophenyl phosphate, 1 mM each phosphoserine and phosphothreonine, 0.2 mM phosphotyrosine, and 100 µM sodium orthovanadate and the protease inhibitors 25 µg each of aprotinin, leupeptin, and pepstatin A per ml and 1 mM phenylmethylsulfonyl fluoride. For Western analyses or immunoprecipitations, the cells were harvested by being scraped into PBS. The pelleted cells were resuspended in p21 buffer (20 mM MOPS (3-[N-morpholino]propanesulfonic acid), 5 mM MgCl2, 0.1 mM EDTA, 200 mM sucrose [pH 7.4]) containing 1% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; U.S. Biochemical Corp.). The cells were lysed for 20 min on ice and centrifuged at 13,000 x g for 10 min, and the supernatant was retained for further experiments. The protein concentration was determined by the method of Bradford (7). For Western analysis of phospho-Bad, cells were harvested and combined with their medium and centrifuged at 1,000 x g for 10 min. The cells were washed once in TBS (Tris-buffered saline; 20 mM Tris, 140 mM NaCl[pH 7.4]) and solubilized in TBS containing 1% NP-40 NP-40 (Igepal; Sigma) and phosphatase and protease inhibitors as described above. After 20 min on ice, the lysate was centrifuged at 13,000 x g, and the supernatant was retained for protein measurements and Western analysis.
Western analysis. Lysates containing equal amounts of protein were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, and following electrophoresis the gel was transferred to a polyvinylidene difluoride membrane (PVDF) (Hybond P; Amersham/Pharmacia Biotech.). Blocking was performed for 45 min using Blocker Casein in PBS (Pierce Chemical Co.) containing 5% newborn calf serum (Life Technologies, Inc.-Invitrogen). The blots were incubated with primary antibodies for 2 to 3 h at room temperature or overnight at 4°C. Following washing in TBS-0.1% Tween, the blots were incubated with secondary antibodies (1:1,000) for 1 h at room temperature. After being washed, the blots were developed, as indicated, by enhanced chemiluminescence (ECL) or ECL-Plus (Amersham-Pharmacia Biotech.) and exposure to film (Hyperfilm ECL; Amersham-Pharmacia Biotech.). The immunoblots were quantitated using a Microtek scanner and NIH Image 1.60b7. The bands were quantitated by normalization with respect to untreated controls.
Immunoprecipitations. JNK was immunoprecipitated from 50 µg of whole-cell lysate by using 1.5 µg of anti-JNK1 polyclonal antibody (Santa Cruz Biotechnology; sc-571) for 1.5 h at 4°C followed by the addition of protein A-Sepharose (Repligen) for 1 h at 4°C. The immunoprecipitates were washed three times with p21 buffer containing 1% CHAPS and then once with p21 buffer prior to the kinase assay. p38 was immunoprecipitated using 3 µl of anti-p38 polyclonal antiserum by the same method as that used for immunoprecipitation of JNK.
Kinase Assays. (i) JNK/p38 assays.
Immunoprecipitates of JNK or p38 were incubated for 20 min at 37°C in kinase buffer consisting of p21 buffer containing protease and phosphatase inhibitors, 10 µM ATP, 5 µg of GST-ATF2 per reaction, and 25 µCi of [
-32P]ATP (6,000 Ci/mmol; New England Nuclear) per reaction. The reactions were terminated by centrifugation at 13,000 x g at 4°C and removal of the supernatant solution. The pellets were washed with p21 buffer, the supernatant solutions were combined, and 5x Laemmli sample buffer was added. The supernatant solutions were loaded onto SDS-12% polyacrylamide gels, and following electrophoresis the gel was transferred to PVDF. Signals were detected using a Molecular Dynamics PhosphorImager.
(ii) Upstream activator double kinase assays.
A 2-µg portion of inactive His6-tagged JNK11 was incubated with 12 µg of untreated or treated cell lysate for 20 min at 30°C in the presence of 50 µM unlabeled ATP, and the His6-tagged JNK was isolated using nickel resin (ProBond; Invitrogen). The isolated JNK was washed with p21 buffer and incubated in kinase buffer with 5 µg of GST-ATF2 at 37°C for 20 min. The supernatant solution was removed, the pellets were washed, and the supernatant solutions were combined, electrophoresed, and transferred as described above. A similar method was used to examine the activation of p38, except that the GST-p38 was isolated after the first kinase reaction with lysate by using glutathione-agarose (Sigma-Aldrich). The second kinase reaction to measure the activity of the isolated GST-p38 was performed as described above using 5 µg of GST-ATF2 (per reaction) as the substrate.
(iii) Akt assays.
Akt assays were performed using an Akt kinase assay kit (Cell Signaling Technology). A 20-µl volume of the anti-Akt slurry was used to immunoprecipitate Akt from 500 µg of cell lysate prepared in the lysis buffer provided in the kit. After being washed, the immunoprecipitates were used in a kinase assay containing ATP and 2 µg (rather than the 1 µg suggested by the manufacturer) of GSK-3ß fusion protein substrate per reaction. The reaction mixtures were incubated for 30 min at 30°C, and the reactions were terminated by the addition of sample buffer. The pellets were washed once with sample buffer, and the supernatants were combined and mixed with Laemmli sample buffer and subjected to SDS-PAGE (12% polyacrylamide). Following transfer to PVDF, the blots were developed with anti-phospho-GSK-3/ß. Bands were visualized using goat anti-mouse antibody-HRP followed by donkey anti-goat antibody-HRP and standard ECL techniques. A nonimmune control immunoprecipitation was performed using mouse IgG and protein G-Sepharose.
Kinase assay products were quantitated using a Microtek scanner and NIH Image 1.60b7. Bands were quantitated by normalization with respect to untreated controls.
Apoptosis assay.
Untreated or treated cells in 12-well cluster plates were scraped in their medium and centrifuged at 500 x g for 5 min. The cell pellet was resuspended in 200 µl of lysis buffer supplied by the manufacturer (Cell Death Detection ELISA Plus kit; Roche Molecular Biochemicals). Then 20-µl aliquots were used in the analysis that measures the appearance and relative amounts of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes), with detection by a microtiter plate reader at 405 nm, as specified by the manufacturer. Incubation was performed overnight at 4°C instead of the 2 to 3 h at room temperature as suggested by the manufacturer. The reading from the negative control (buffer only) supplied by the manufacturer was subtracted from all sample values. All experiments were performed in triplicate, and error bars indicate the sample standard deviation of the mean (
n - 1).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and cycloheximide (Fig. 1B). We have found little (<5%) difference in the apoptosis measurements over the 4-h treatment period between untreated and cycloheximide-only-treated cells (reference 129 and data not shown). The N-Ras knockout cells underwent a five- to sixfold increase in apoptosis after 4 h of treatment with TNF- plus cycloheximide. Since the N-Ras knockout cells possess very low levels of pBad even at steady state, treatment with LY294002 does not cause a further increase in their level of apoptosis in response to TNF- and cycloheximide. These data suggested that c-N-Ras may be providing more than one survival signal and that one of its survival functions is Akt independent.
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per ml in the presence of cycloheximide. All of the cell lines expressed minimal levels of phosphorylated JNK (pJNK) at steady state (t = 0). The level of pJNK in the N-Ras knockout cells increased by 15 min, peaked at 1 h, and remained elevated by 5 h after treatment with TNF- plus cycloheximide. Treatment of N-Ras knockout cells with low doses of TNF- in the presence of cycloheximide for 4 to 5 h is sufficient to cause approximately 50% cell death by apoptosis (129). The level of pJNK in control N+/+ cells was moderately increased, peaking at 1 h (Fig. 6B and data not shown) and then declining to basal levels by 3 to 5 h after treatment with TNF- plus cycloheximide. c-N-Ras reconstituted knockout cells, which express c-N-Ras at control cell levels, possessed levels of pJNK that approximated those seen in control cells, although they were not completely down to basal levels by 5 h after treatment with TNF- plus cycloheximide. This may correlate with their ability to only partially rescue the heightened apoptotic sensitivity of the parental knockout cells in response to TNF- plus cycloheximide (129). The differences observed in pJNK are not a result of changes in the total abundance of JNK1 protein. All of the cell lines possessed similar levels of total JNK1 in the absence or presence of TNF- plus cycloheximide (Fig. 2B). Similar results have been observed using other independently derived N-Ras knockout and control cell lines as well as other clones of c-N-Ras reconstituted cell lines (data not shown).
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per ml in the presence of cycloheximide. At the indicated times, all of the cells were harvested and JNK1 was immunoprecipitated from the lysates for measurement of its in vitro kinase activity. JNK activity in the N-Ras knockout cells increased approximately 14-fold and remained elevated as these cells underwent apoptosis (Fig. 2C). Control N+/+ cells exhibited a 4.5-fold increase in JNK activity that was transient in nature. The pattern of JNK activity in the c-N-Ras reconstituted cells more closely resembled that observed in the control N+/+ cells in that it was also transiently increased. Both reconstituted cell lines, however, exhibited a larger increase in JNK activity and were more delayed in returning to baseline than the control cells. As observed with the phosphorylation of JNK, this may correlate with the ability of the ectopically expressed c-N-Ras to only partially rescue the heightened apoptotic sensitivity of the parental knockout cells in response to TNF- plus cycloheximide (129). Nevertheless, these data demonstrate that in our system, a robust and sustained activation of JNK correlates with apoptosis. The data also suggest that attenuation of the JNK response to apoptotic induction may be another mechanism by which c-N-Ras provides a cellular survival function.
We sought to determine whether the patterns of JNK phosphorylation and activity observed in the N-Ras knockout and control cells were similar by using other methods of apoptotic induction. If this were the case, it would suggest that the role of c-N-Ras in cell survival in response to apoptotic induction is global and is not restricted to a single type of stimulus. Fas is another member of the TNF receptor family which can trigger apoptosis in some cell types (1, 17, 104, 111, 123). We previously demonstrated that the N-Ras knockout fibroblasts are more sensitive to activation of the Fas receptor than are control N+/+ fibroblasts or c-N-Ras reconstituted cells (129). As observed with TNF- plus cycloheximide, N-Ras knockout cells demonstrated a strong and sustained stimulation of JNK phosphorylation in response to Fas ligation compared to untreated cells. Control N+/+ cells did not show any significant increases in pJNK in response to anti-Fas treatment (Fig. 2D). This correlates with their failure to undergo apoptosis under these conditions (129). We observed significantly less pronounced activation of JNK in the c-N-Ras reconstituted cells under these conditions (Fig. 2D). This also correlates with the ability of ectopically expressed c-N-Ras to provide protection from Fas-mediated apoptosis in the N-Ras knockout cells (129). Differences in JNK phosphorylation did not arise from different levels of total JNK protein abundance. N-Ras knockout, control, and c-N-Ras reconstituted cells contained similar amounts of JNK1 protein in the presence or absence of anti-Fas antibody (data not shown). These results are consistent with those obtained using other independently isolated N-Ras knockout and control cell lines (data not shown). We measured JNK activity from untreated or anti-Fas treated cells by an in vitro kinase assay. We observed a slower increase in JNK activity than in phosphorylation of JNK in the N-Ras knockout cells; however, the JNK activity remained elevated as these cells underwent apoptosis (Fig. 2E). Similar to the levels of phosphorylated JNK, the c-N-Ras reconstituted knockout cells displayed less pronounced and delayed JNK activity upon anti-Fas treatment, with the control cells displaying little JNK activity during this time course. This correlates with their higher resistance to the induction of apoptosis. These data again parallel the results observed with TNF- treatment. The activation of JNK in response to serum withdrawal also parallels the results obtained with TNF- and Fas stimulation, although the time course is slightly different (data not shown).
We tested the specificity of the apparent regulation of JNK activation by c-N-Ras. We previously demonstrated that ectopic overexpression of c-K(A)-Ras in the N-Ras knockout cells could not substitute for wild-type levels of c-N-Ras in providing protection from TNF--plus-cycloheximide-induced apoptosis (129). The stable c-K(A)-Ras-expressing N-Ras knockout cells contain higher levels of c-K(A)-Ras than do the parental N-Ras knockout cells or control N+/+ cells (129). These c-K(A)-Ras-expressing cells and the parental N-Ras knockout cells and control N+/+ cells were treated with TNF- plus cycloheximide, and the levels of pJNK were examined by Western analysis. Two different c-K(A)-Ras-expressing cell lines demonstrated sustained phosphorylation of JNK similar to that observed for the parental N-Ras knockout cells (Fig. 2F). Control cells again showed reduced and transient JNK phosphorylation under these conditions. The total abundance of JNK1 protein was unchanged between the cell lines used and under the treatment conditions employed (data not shown). Similar to c-K(A)-Ras, c-K(B)-Ras expression in the N-Ras knockout cells does not lead to a reduction in JNK phosphorylation in response to treatment with TNF- plus cycloheximide (data not shown). Taken together, these data provide evidence that c-N-Ras generates a second survival signal in addition to up-regulation of steady-state pAkt and pBad levels. In this case, c-N-Ras specifically appears to attenuate the magnitude and duration of JNK activation in response to the induction of apoptosis.
c-N-Ras also attenuates the activation of p38 in response to apoptotic induction.
Prolonged activation of p38 has been associated with apoptosis in some cell types (33, 52, 62, 63, 131, 135). We examined the activation of p38 in N-Ras knockout and control cells in response to apoptotic induction. N-Ras knockout cells treated with low-dose TNF- in the presence of cycloheximide possessed elevated and sustained phosphorylated p38 levels (Fig. 3A). We observed only minimal phosphorylation of p38 in control N+/+ cells. c-N-Ras reconstituted cells exhibited a minor basal phosphorylation of p38 that increased only a little at 1 h and declined thereafter. These c-N-Ras reconstituted cells are partially protected from TNF--induced apoptosis (data not shown) compared to parental N-Ras knockout cells, similar to that observed with other c-N-Ras reconstituted cell lines (129). Measurements of p38 activity correlated with the results obtained by analysis of the phosphorylation of p38. N-Ras knockout cells exhibited increasing p38 kinase activity following treatment with TNF- plus cycloheximide, which was not observed in either control N+/+ cells or c-N-Ras reconstituted cells (Fig. 3B). We observed only transient p38 kinase activity that decreased by approximately 30 and 50% in control cells and the c-N-Ras reconstituted cells, respectively, 4 h after treatment with TNF- plus cycloheximide. Similar to the results with JNK, the total amount of p38 protein was unchanged in all the cell lines both at steady state (t = 0) and under the treatment conditions used (Fig. 3C). This suggests that the differences observed between N-Ras knockout and control cells in p38 phosphorylation and activity do not arise from changes in p38 protein levels. We have also observed elevated and prolonged p38 phosphorylation and activity in the N-Ras knockout cells in response to Fas activation. In response to Fas ligation, activation of p38 is minimal in control cells and only transiently increased in c-N-Ras reconstituted knockout cells (data not shown).
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(Fig. 3A) and Fas (data not shown), serum deprivation induced an elevated and sustained phosphorylation of p38 that was not observed in either control N+/+ cells or the c-N-Ras reconstituted cells (Fig. 3D). The total amount of p38 protein was unchanged between N-Ras knockout and control cells up to 48 h of serum starvation (data not shown). These data support a functional role for c-N-Ras in cell survival through attenuation of the duration and, in some cases, the magnitude of both JNK and p38 activation following apoptotic induction by several different experimental conditions. One question remaining is the mechanism through which c-N-Ras controls the duration of JNK and p38 activation. It is possible that c-N-Ras either downregulates upstream JNK/p38 activators or increases the activity of JNK/p38-specific phosphatases or some combination of the two mechanisms.
c-N-Ras downregulates the activation of upstream kinase activators of JNK and p38.
We have previously demonstrated that c-N-Ras regulates the level of pBad and that this regulation appears to be dependent on Akt activity (129). One of the major questions in the present study is the nature of the mechanism by which c-N-Ras regulates or attenuates the activation of JNK or p38. We investigated the activation of upstream kinase activators of JNK and p38 in N-Ras knockout and control N+/+ cells by using phosphospecific antibodies to these upstream kinase activators. MKK4 is one of the upstream activators of JNK (21, 48, 49, 75). In response to treatment with TNF- plus cycloheximide, a sustained elevation of the level of phosphorylated MKK4 was observed in the N-Ras knockout cells (Fig. 4A, upper panel). Control N+/+ cells had a basal level of phosphorylated MKK4 that increased slightly by 1 h and was reduced by 3.5 h after treatment with TNF- plus cycloheximide. We have found that the steady-state (t = 0) level of pMKK4 in the control cells is subject to variability; however, it is always reduced to minimal levels by 3 to 4 h after treatment with TNF- plus cycloheximide (data not shown). We observed only transiently increased levels of phosphorylated MKK4 in the c-N-Ras reconstituted cells. The levels of total MKK4 protein were constant in all the cell lines both at steady state and following TNF- treatment (Fig. 4A, lower panel). These data suggest that one mechanism by which c-N-Ras regulates JNK activation may be through regulation of MKK4 activity.
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per ml in the presence of cycloheximide led to a sustained increase in the phosphorylation of MKK3/6 (Fig. 4B). In contrast, we observed only a transient increase in the level of phosphorylated MKK3/6 in the N+/+ control cells, and the level was completely down to basal levels by 3.5 h posttreatment. c-N-Ras reconstituted cells possessed levels of phosphorylated MKK3/6 that peaked at 1 h and declined by 3.5 h after treatment with TNF- plus cycloheximide (Fig. 4B, upper panel). The levels of total MKK3 protein were similar in all the cell lines and at all the time points examined (Fig. 4B, lower panel). These data suggest that, as was observed with JNK activity, attenuation of p38 activity by c-N-Ras correlates with a decrease in the duration of the activation of the upstream kinase activators of p38. Unfortunately, we have been unable to specifically immunoprecipitate any of the MKK proteins to test for changes in their relative specific activities. These experiments await the production of antibodies that will selectively immunoprecipitate the various MKK proteins for in vitro kinase assays. Even with the available reagents, however, we consistently observed phosphorylated MKK4 and MKK3/6 at the 3.5-h time point in the N-Ras knockout cells, in contrast to the lack of signal in the N+/+ control cells.
To validate the results obtained by analyzing the phosphorylation of MKK4 and MKK3/6, we used unactive recombinant JNK and p38 in in vitro double kinase assays. These experiments allowed us to analyze the generic activity of the upstream kinase activators of JNK and p38 by using recombinant JNK and p38 as substrates for in vitro kinase assays. N-Ras knockout fibroblasts, control N+/+ cells, and c-N-Ras reconstituted cells were left untreated or treated with TNF- plus cycloheximide and then used for lysate preparation. Unactive recombinant, His6-tagged JNK was mixed with an aliquot of each lysate and incubated in the presence of ATP. Following this first kinase reaction, the recombinant JNK was isolated using a nickel resin and incubated with [-32P]ATP and purified GST-ATF2. The amount of phosphorylated GST-ATF2 was quantitated by SDS-PAGE and PhosphorImager analysis. As observed by examination of the phosphorylation state of MKK4, there was increased and persistent activation of upstream JNK activators in the N-Ras knockout cells. Similarly, as was seen with endogenous JNK activity, there was elevated and sustained activation of the recombinant JNK incubated with lysates from the N-Ras knockout cells (Fig. 4C). Both the control N+/+ cells and the c-N-Ras reconstituted knockout cells exhibited only transient activation of the recombinant JNK, indicating only transient activation of the upstream kinase activators of JNK in these cells that contain c-N-Ras.
A similar experiment was performed using unactive recombinant GST-tagged p38. Aliquots of lysates from untreated or TNF--plus-cycloheximide-treated cells were incubated with ATP and recombinant p38. The p38 protein was isolated using glutathione-agarose and incubated with [-32P]ATP and GST-ATF2. PhosphorImager analysis revealed a similar pattern of activation of the recombinant p38 (Fig. 4D) to that observed with recombinant JNK (Fig. 4C) or endogenous p38 (Fig. 3A). The results reflected similar differences in upstream kinase activity to those seen in the analysis of MKK3/6 phosphorylation (Fig. 4B). There was elevated and sustained activation of one or more specific upstream kinase activators of p38 in the N-Ras knockout cells compared to both control N+/+ cells and c-N-Ras reconstituted cells (Fig. 4D). The data from these generic double-kinase assays more closely resemble the data obtained with the endogenous JNK/p38 immunoprecipitation and direct kinase assays than with the phosphoblots of MKK4 and MKK3/6. This might suggest that there are other JNK or p38 upstream activators that are also downregulated through a c-N-Ras-specific function.
We next tested whether N-Ras knockout cells were able to inactivate JNK in a context outside of apoptosis. It was possible that these cells lacked a phosphatase, preventing the down-regulation of JNK. To test this possibility, we treated cells with a mitogen that does not cause apoptosis. Cells were untreated in normal medium containing serum (to prevent serum withdrawal-dependent induction of apoptosis in the N-Ras knockout cells) for 48 h and then stimulated for various times with 10 ng of epidermal growth factor per ml (EGF). At the indicated times, the cells were harvested and lysates were prepared to assess the level of phosphorylated JNK and ERK. The level of phosphorylated JNK peaked at 15 min and decreased thereafter in all of the cell lines. Similar results were observed with pERK, except that its level remained elevated at 1 h following EGF treatment and then declined (Fig. 4E). There were no significant differences in total JNK1 or total ERK2 between N-Ras knockout, control, and c-N-Ras reconstituted cells, which were unchanged in response to EGF treatment (data not shown). These data imply that the N-Ras knockout cells are fully capable of downregulating activated JNK. Furthermore, the data suggest that activation of JNK occurs in a transient fashion in response to mitogen stimulation, unlike the sustained activation observed in the N-Ras knockout cells following induction of apoptosis (Fig. 2A and B).
JNK plays a proapoptotic role in the N-Ras knockout and control cells. Our data suggest that persistent JNK activity, and possibly p38 activity, correlates with an apoptotically sensitive phenotype. We generated stable cell lines expressing an estrogen-regulated MEKK3 construct in an effort to determine whether persistent JNK activity is either proapoptotic or a failed protective mechanism. As a positive control for pJNK, the N-Ras knockout and control cells were treated for 1 h with 20 µg of anisomycin per ml. ß-Estradiol did not activate JNK in the untransfected N-Ras knockout or control N+/+ cell lines (Fig. 5A). There was a modest increase in the level of phosphorylated JNK upon ß-estradiol treatment of the MEKK3:ER-transfected cell lines, demonstrating that the MEKK3:ER was performing as expected. The increased JNK phosphorylation persisted over the time course of the experiments with continuous exposure to ß-estradiol (data not shown). There were no significant differences in total JNK1 protein between the MEKK3:ER-transfected cell lines and either parental control cells or N-Ras knockout cells, and the JNK1 protein levels were unchanged by ß-estradiol treatment (data not shown). There may be a small amount of leakiness of the MEKK3:ER construct, since the basal levels of pJNK in the transfected cell lines were slightly elevated compared to that observed in the parental cell lines (Fig. 5A).
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plus cycloheximide in the presence or absence of ß-estradiol. As expected, there was a five- to sixfold increase in apoptosis after 4 h of treatment with TNF- in the N-Ras knockout cells (Fig. 5B). ß-Estradiol treatment alone did not result in increased apoptosis in the untreated N-Ras knockout cells. Similarly, ß-estradiol did not enhance their sensitivity to TNF--induced apoptosis. The control N+/+ cells remained relatively insensitive to TNF- plus cycloheximide in the absence or presence of ß-estradiol. ß-Estradiol treatment alone did not lead to a substantial increase in apoptosis in either the N-Ras knockout or control cells stably expressing the MEKK3:ER construct (Fig. 5B). However, the level of apoptosis in the MEKK3:ER-transfected control cell lines was significantly increased in response to combined treatment with ß-estradiol and TNF- plus cycloheximide (Fig. 5B). Again, continuous treatment with ß-estradiol in the MEKK3:ER-transfected cells lead to sustained phosphorylation of JNK (data not shown). Therefore, the results observed with the control N+/+ cells stably expressing MEKK3:ER demonstrate that sustained activation of JNK plays a proapoptotic role. These data verify our interpretation of the results obtained with the N-Ras knockout cells, suggesting that sustained JNK activation can play a proapoptotic role.
c-N-Ras regulates the duration of JNK activation through a distinct set of downstream targets.
In addition to examining which upstream JNK activators participate in sustained JNK activation, we examined which N-Ras-dependent pathway(s) might contribute to the attenuation of JNK activation. GTP-bound Ras can bind many different effectors (69, 106). Specific mutations within the switch 1 region of the effector-binding domain of Ras were found to produce Ras proteins capable of binding a limited number of effectors (125). The T-to-S mutation at amino acid 35 (35S) produces a Ras protein capable of binding only Raf-1. The Y40-to-C mutation (40C) renders the Ras-GTP protein capable of binding only PI3-kinase (p110 and ß). The E37-to-G mutation (37G) produces a Ras protein capable of binding only RalGDS (125). Since these mutants were first described, the 37G mutant Ras protein has also been shown to bind Rin 1, AF-6, PLC, and p110
(18, 54, 60, 106). The failure of each of these Ras switch 1 mutations in a G12V background to transform fibroblasts suggests that Ras must activate more than one effector pathway to generate a transformed phenotype (125, 126). For the present study, the same effector domain mutations were made in a Q61K-N-Ras background. We chose to use the selective switch 1 mutants in an activated (K61) background to overcome any potential problems that might arise in the interaction of the cellular switch 1 mutants with Ras exchange factors. These mutant N-Ras proteins were transfected into N-Ras knockout cells, and stable clones were selected in G418. The level of effector domain mutant N-Ras expression was compared to the level of endogenous c-N-Ras in the control N+/+ cells (Fig. 6A).
We tested the switch 1 N-Ras-expressing cells for the predicted interactions with the appropriate downstream effectors. We first examined the ability of the 35S (Raf-1-binding) N-Ras mutants to lead to elevated steady-state Erk/mitogen-activated protein (MAP) kinase phosphorylation. N-Ras knockout and control N+/+ cells and several cell lines expressing the switch 1 mutant N-Ras proteins were subcultured in complete serum-containing medium and left untreated for 48 h. The cells were then harvested, and the level of phospho-Erk (pErk) was examined by immunoblotting. There was elevated pErk in both 35S N-Ras-expressing cell lines compared to N-Ras knockout, control N+/+, or the other switch 1 mutant-expressing cells (37G or 40C) (Fig. 6B, upper panel). The levels of total Erk protein were the same in all the cell lines examined (Fig. 6B, lower panel). We chose to verify these results by further testing the function of the 35S mutant N-Ras proteins after stable expression in NIH 3T3 cells. Stable cell lines expressing the various switch 1 mutant N-Ras proteins (Fig. 6C, upper panel) were grown in complete medium and harvested after reaching approximately 80% confluence, as described in Materials and Methods. The levels of pErk were examined by immunoblotting and compared to those in vector-transfected cells (Fig. 6C, lower panel). The results demonstrated that the level of pErk was elevated in the 35S (Raf-1-binding) N-Ras-expressing cells compared to either the 37G or the 40C mutant N-Ras-expressing cells (Fig. 6C, lower panel). The levels of expression of total Erk1 protein were identical in all the cell lines examined (data not shown).
We next tested the ability of the 40C (PI3-kinase-binding, p110/ß) mutant N-Ras-expressing cells to lead to elevated Akt activity since Akt is activated by PI3-kinase. N-Ras knockout cells, control N+/+ cells, and N-Ras knockout cells stably expressing the various switch 1 mutant N-Ras proteins were grown in complete medium for 48 h and harvested for an Akt assay by using the detergent-containing buffer supplied by the manufacturer in the Akt kinase assay kit, as described in Materials and Methods. Akt was immunoprecipitated from equal amounts of protein (0.5 mg) and used in a kinase assay with GSK-3ß fusion protein as the substrate. Following electrophoresis and transfer to PVDF, the blot was developed with anti-phospho-GSK-3/ß. There was elevated steady-state Akt activity in both 40C mutant N-Ras-expressing cell lines (Fig. 6D). Similarly, there was increased steady-state Akt activity in the control cells, which compares favorably with previous results demonstrating that the control cells possess elevated phospho-Akt levels at steady state (129). The levels of total Akt were similar in all the cell lines tested (data not shown). Finally, all of the cell lines were tested using a Ral-binding domain protein to pull down Ral-GTP as a test for the function of the 37G (RalGDS, etc., binding) N-Ras mutant. Unfortunately, there was a high level of Ral-GTP in all of the cell lines including the N-Ras knockout and control cells, making it difficult to distinguish small differences resulting from expression of the 37G mutant (data not shown). All of the cell lines expressed equal amounts of both RalA and RalB (data not shown). The pulldown assay is inherently inadequate because any Ral-GTP that is bound to effector proteins would be left behind in this type of pulldown assay. Furthermore, this assay also tests the ability of the 37G mutant to bind only to RalGDS and none of the other effectors to which this mutant is reported to bind. We have, however, found that there was modestly elevated Akt activity in the 37G-expressing cells compared to either the parental N-Ras knockout cells or the 35S-expressing cells (Fig. 6D). Since the 37G mutant also binds the p110 isoform of PI3-kinase, these data suggest that this mutant is also functional in the N-Ras knockout cells.
We sought to investigate whether expression of any of the effector domain mutant N-Ras proteins leads to attenuation of JNK activation, as observed in control or c-N-Ras reconstituted cells. N-Ras knockout cells, control N+/+ cells, c-N-Ras reconstituted cells, and N-Ras knockout cells expressing the effector domain mutant N-Ras proteins were left untreated or treated with TNF- and cycloheximide as described above. At the indicated times, all the cells were harvested and lysates were prepared for the analysis of JNK phosphorylation. Only the 37G N-Ras protein was capable of restoring the attenuation of JNK activation, as observed with control N+/+ cells (Fig. 7A).
The 37G mutant N-Ras-expressing cells demonstrated a transient increase in JNK phosphorylation, as observed with control N+/+ or c-N-Ras reconstituted cells. The 37G mutant N-Ras was as effective as wild-type c-N-Ras in restoring JNK regulation. Neither the Raf-1-binding 35S nor the PI3-kinase-binding 40C mutant N-Ras proteins were capable of restoring JNK regulation. The total levels of JNK1 protein were similar in all cell lines and under the treatment conditions used (data not shown). These data imply that attenuation of JNK activation by N-Ras does not proceed through either a Raf-1- or PI3-kinase (p110 or p110ß)-dependent pathway. The data do suggest that the attenuation of JNK activation by N-Ras during apoptotic induction proceeds through a distinct set of downstream targets, which may include RalGDS, AF-6, Rin 1, PLC, and/or p110.
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domain and the N-terminal transactivation domain (2, 23, 90, 94, 107, 108). Cells were treated with TNF- and cycloheximide as described above. The level of Ser63 phosphorylation of endogenous c-Jun was analyzed using a phospho-specific antibody. We observed a large increase in the level of Ser63 phosphorylated c-Jun 4 h after treatment with TNF- plus cycloheximide in the N-Ras knockout cells (Fig. 7B). This is a point where these cells are nearly 50% dead by apoptosis (129). The level of Ser63 c-Jun phosphorylation was only minimally elevated in the c-N-Ras reconstituted cells. Similar to the N-Ras knockout parental cells, N-Ras knockout cells stably expressing either the 35S (Raf-1-binding) or 40C (PI3-kinase-binding) switch 1 mutant N-Ras proteins exhibited elevated and sustained Ser63 c-Jun phosphorylation. We observed identical results using other clones of N-Ras knockout cells expressing either the 35S or 40C mutant N-Ras proteins (data not shown). In sharp contrast, little Ser63 c-Jun phosphorylation was observed in either clone of the N-Ras knockout cells expressing the 37G (RalGDS) effector domain mutant N-Ras protein, similar to that observed with control N+/+ cells. Similar results were observed when the Ser73 c-Jun phosphorylation site was examined (data not shown). The differences in c-Jun phosphorylation were not a result of different levels of total c-Jun protein. All of the cell lines possessed similar levels of total c-Jun protein in the presence or absence of apoptotic treatments (data not shown). These data strongly suggest that attenuation of JNK activation by c-N-Ras proceeds through a mechanism that is independent of both Raf-1 and PI3-kinase (p110 or p110ß).
Because the 37G switch 1 mutant N-Ras protein expressed in the N-Ras knockout cells restores JNK regulation, similar to endogenous wild-type c-N-Ras, we determined whether this mutant could also rescue the apoptotically sensitive phenotype of the parental N-Ras knockout cells. N-Ras knockout cells, control N+/+ cells, c-N-Ras restored cells, and N-Ras knockout cells expressing different levels of the effector domain mutant N-Ras proteins were left untreated or treated with TNF- and cycloheximide as described above. After 4 h the cells were harvested and the level of apoptosis was measured using a Cell Death Detection ELISA. The N-Ras knockout cells underwent an approximately fourfold increase in apoptosis compared to untreated cells (untreated, 0.2; treated, 0.77 [data not shown and Fig. 7C]). The control cells and the c-N-Ras reconstituted cells did not undergo significant apoptosis under these conditions. Untreated values were approximately 0.3 for the control N+/+ cells, c-N-Ras reconstituted cells, and the N-Ras knockout cells expressing the various effector domain mutant N-Ras constructs (data not shown). N-Ras knockout cells expressing either the Raf-1-selective 35S or the PI3-kinase-selective 40C mutant N-Ras proteins were not protected from undergoing TNF--dependent apoptosis (Fig. 7C). As observed with JNK activation, expression of the 37G mutant N-Ras protein completely reversed the apoptotic sensitivity of the parental N-Ras knockout cells. This reversion by the 37G mutant is quite significant since it occurs at much lower levels of expression (clone 4) than the levels of expression of the 35S or the 40C mutant N-Ras proteins, which are ineffective in rescuing the N-Ras knockout cells from apoptosis. Taken together, these results suggest that attenuation of JNK activation and protection from apoptosis by N-Ras occurs through a mechanism that is independent of signaling through either Raf-1 or PI3-kinase (p110 or p110ß). Since the 37G mutant N-Ras protein is capable of binding RalGDS, Rin 1, AF-6, PLC, and p110, additional studies are be needed to examine the contribution of each pathway, RalGDS, Rin 1, PLC, or p110 to the regulation of JNK activation and protection from apoptosis. Studies with PI-3 kinase inhibitors suggest that p110 does not contribute to the attenuation of JNK activation (data not shown) (see Discussion).
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= 405 nm. Assays were performed in triplicate, and the results are plotted with error bars representing the sample standard deviation of the mean (