Endogenous c-N-Ras Provides a Steady-state Anti-apoptotic Signal
2000; Elsevier BV; Volume: 275; Issue: 25 Linguagem: Inglês
10.1074/jbc.m000250200
ISSN1083-351X
AutoresJanice C. Wolfman, Alan Wolfman,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoWe report that c-N-Ras possesses an isoform-specific, functional role in cell survival under steady-state conditions. This function includes protection from programmed cell death by serum deprivation or upon treatment with apoptosis-inducing agents. The data demonstrate that c-N-Ras may play a functional role in the regulation of steady-state phosphorylated Akt and serine 136-phosphorylated Bad (Ser136-pBad). Immortalized N-Ras knockout fibroblasts possess nearly undetectable levels of steady-state Ser136-pBad. In contrast, wild-type control cells and the N-Ras knockout cells ectopically expressing c-N-Ras at control levels maintained easily detectable levels of Ser136-pBad both at steady-state and following treatment with tumor necrosis factor α. Similar results were seen with Ser112-pBad. These differences did not arise from differences in total Bad protein levels. These data correlate with the observation that the N-Ras knockout cells exhibit a heightened susceptibility to the induction of apoptosis. Ectopic expression of c-N-Ras in the N-Ras knockout cells at endogenous levels, compared with control cells, significantly rescues the apoptotically sensitive phenotype. Elevated expression of either c-Kirsten A-Ras or c-Kirsten B-Ras did not reverse the apoptotic sensitivity of the N-Ras knockout cells or result in increased levels of either phospho-Akt or phospho-Bad. Our results indicate that, at steady state, c-N-Ras possesses an isoform-specific, functional role in cell survival. We report that c-N-Ras possesses an isoform-specific, functional role in cell survival under steady-state conditions. This function includes protection from programmed cell death by serum deprivation or upon treatment with apoptosis-inducing agents. The data demonstrate that c-N-Ras may play a functional role in the regulation of steady-state phosphorylated Akt and serine 136-phosphorylated Bad (Ser136-pBad). Immortalized N-Ras knockout fibroblasts possess nearly undetectable levels of steady-state Ser136-pBad. In contrast, wild-type control cells and the N-Ras knockout cells ectopically expressing c-N-Ras at control levels maintained easily detectable levels of Ser136-pBad both at steady-state and following treatment with tumor necrosis factor α. Similar results were seen with Ser112-pBad. These differences did not arise from differences in total Bad protein levels. These data correlate with the observation that the N-Ras knockout cells exhibit a heightened susceptibility to the induction of apoptosis. Ectopic expression of c-N-Ras in the N-Ras knockout cells at endogenous levels, compared with control cells, significantly rescues the apoptotically sensitive phenotype. Elevated expression of either c-Kirsten A-Ras or c-Kirsten B-Ras did not reverse the apoptotic sensitivity of the N-Ras knockout cells or result in increased levels of either phospho-Akt or phospho-Bad. Our results indicate that, at steady state, c-N-Ras possesses an isoform-specific, functional role in cell survival. Harvey Kirsten A and B, respectively phosphatidylinositol serine 473-phosphorylated Akt phosphorylated Bad serine 136-phosphorylated Bad serine 112-phosphorylated Bad extracellular signal-regulated kinase MAP kinase-ERK1 mitogen-activated protein mitogen-activated protein kinase horseradish peroxidase mouse embryo fibroblast phosphate-buffered saline N-Ras knockout cells N-Ras heterozygous cells control cells N-Ras knockout cells ectopically expressing c-N-Ras at control cell levels K-Ras knockout cells control cells, MOPS, 3-(N-morpholino)propanesulfonic acid 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid Tris-buffered saline polyvinylidene difluoride bromo-dUTP nick end labeling enzyme-linked immunosorbent assay tumor necrosis factor α There are four mammalian Ras isoforms: Harvey (Ha),1 N, and two splice variants of the Kirsten gene, Kirsten A (K(A)) and Kirsten B (K(B)). All four proteins are highly homologous except for the C terminus, where they share no sequence similarity. Ras GTP, the active form, interacts with diverse targets within the cell. Amino acids 32–40 and 60–72 comprise the switch 1 and switch 2 regions, respectively, which are identical in all isoforms (1.Wittinghofer A. Pai E.F. Trends Biochem. Sci. 1991; 16: 382-387Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 2.Ma J.P. Karplus M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11905-11910Crossref PubMed Scopus (148) Google Scholar). When Ras binds GTP, both regions undergo conformational changes to form the effector binding pocket (3.Malumbres M. Pellicer A. Front. Biosci. 1998; 3: 887-912Crossref PubMed Scopus (10) Google Scholar). Distinct Ras isoform functions are now becoming apparent. Transformation of C3H10T1/2 fibroblasts by expression of oncogenic G12V-Ha-Ras at endogenous levels requires the cooperation with cellular N-Ras (4.Hamilton M. Wolfman A. 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Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2233) Google Scholar, 35.Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3224) Google Scholar), positioning Akt function in the cell survival pathway. Ras has been reported to have a functional role in many cellular processes including cell proliferation, migration, differentiation, apoptosis, and certain immune responses (18.Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (918) Google Scholar, 36.Downward J. Curr. Opin. Genet. Dev. 1998; 8: 49-54Crossref PubMed Scopus (503) Google Scholar). Apoptosis, also known as programmed cell death, is an ordered disassembly of a cell, characterized by specific cellular and phenotypic changes including cell shrinkage, membrane blebbing, and DNA degradation (37.Dragovich T. Rudin C.M. Thompson C.B. 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This antiapoptotic signal appears to be generated, at least in part, through regulation of basal phospho-Bad levels. Neither c-K(A)- nor c-K(B)-Ras can substitute for this c-N-Ras survival function. Bad polyclonal, phosphospecific Bad polyclonal (Ser112 and Ser136), Akt polyclonal, and Ser473 phospho-Akt polyclonal antibodies were from New England Biolabs. Phospho-MAP kinase monoclonal, anti-N-Ras monoclonal, anti-ERK2 polyclonal, anti-K(A)-Ras polyclonal, and anti-K(B)-Ras polyclonal antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-FLAG monoclonal antibody was from Eastman Kodak Co. Hamster anti-mouse Fas receptor antibody (clone Jo2) (for activation of the Fas receptor) was from Pharmingen (San Diego, CA). Anti-Fas/CD95 antibody (used for Western analysis of Fas receptor) was from Transduction Laboratories. Anti-p55 TNF receptor I was from Biodesign International. Anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP) was from Transduction Laboratories, and goat anti-mouse-HRP was from Kirkegaard and Perry Laboratories (Gaithersburg, MD). N-Ras knockout (N−/−), heterozygote (N+/−), and control N+/+ mouse embryo fibroblasts (MEFs) were a generous gift from R. Kucherlapati (Albert Einstein College of Medicine) (47.Umanoff H. Edelmann W. Pellicer A. Kucherlapati R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1709-1713Crossref PubMed Scopus (204) Google Scholar). K-Ras knockout and control K+/+ MEFs were a generous gift from T. Jacks (Howard Hughes Medical Institute, Massachusetts Institute of Technology) (48.Johnson L. Greenbaum D. Cichowski K. Mercer K. Murphy E. Schmitt E. Bronson R.T. Umanoff H. Edelmann W. Kucherlapati R. Jacks T. Genes Dev. 1997; 11: 2468-2481Crossref PubMed Scopus (432) Google Scholar). MEFs were immortalized by a modification of the 3T3 protocol (49.Todaro G.J. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (1984) Google Scholar). The MEFs were passaged 1:3 every 7 days until they developed a fibroblast morphology. To avoid any cell-specific changes arising from immortalization, multiple, independently isolated cell lines were used throughout these studies. Cells were grown in complete medium consisting of Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal bovine serum (Atlanta Biologicals), 1× nonessential amino acids, and 1× penicillin/streptomycin (Life Technologies). Cells were kept in complete medium in all experiments unless otherwise stated. MEFs were grown in complete medium with additional serum to a final concentration of 20%. Serum starvation was performed by rinsing cells twice with phosphate-buffered saline (PBS; 20 mm Na2HPO4, 120 mmNaCl, pH 7.4) and incubation in Dulbecco's modified Eagle's medium containing nonessential amino acids and penicillin/streptomycin. Recombinant murine TNFα (Calbiochem) was dissolved in 0.2-μm filtered PBS containing 0.1% bovine serum albumin (Sigma) and stored in aliquots at −80 °C. We have found that the TNFα potency varied with the number of freeze/thaw cycles. In general, each aliquot was used only twice. Activation of the Fas receptor was achieved by incubation of cells for the times indicated in complete medium containing 1 μg/ml murine anti-Fas receptor (Pharmingen, clone Jo2, form NA/LE) and 0.5 μg/ml recombinant protein G (Sigma). Staurosporine (Sigma) was dissolved in Me2SO and used at 75–100 nm. N-Ras knockout cells stably expressing wild-type c-N-Ras (N−/−wtN cell lines) were generated by transfection of N−/− cells using Lipofectamine Plus (Life Technologies) with c-N-Ras/pIBW3 (a gift from Angel Pellicer, New York University), which has the c-N-Ras gene under the control of the thymidine kinase promoter, and selection in G418 (Fisher). Stable clones were maintained in complete medium containing 200 μg/ml G418. N-Ras knockout cells stably expressing Bcl-2-FLAG (a gift from Alex Almasan, Cleveland Clinic Foundation) were generated by the same protocol. K(A)-Ras was cloned by polymerase chain reaction (Expand High Fidelity PCR System; Roche Molecular Biochemicals) from a bacterial expression vector containing the sequence of c-K(A)-Ras (gift from Berthe Willumsen, University of Copenhagen). Primers corresponding to the N-terminal region of c-K(A)-Ras (forward, 5′-AAGCTTCCCGGGGCGGCCGCGGATCCATGACGGAAT-3′) and the reverse complement of the C-terminal region of c-K(A)-Ras (reverse, 5′-ATCGATGTCGACGAGCTCTCTAGATTACATTATAACGCATTT-3′) were prepared by Life Technologies, Inc. Following the polymerase chain reaction, the product was ligated into pTargeT (Promega) containing a cytomegalovirus enhancer and promoter and the ligation product used to transform JM109-competent Escherichia coli cells. Colonies were selected on LB plates containing 100 μg/ml ampicillin (U.S. Biochemical Corp.) and screened for the presence and direction of the transgene by restriction digest. Positive, forward clones were used to transfect N-Ras knockout fibroblasts by the method described above. A similar procedure was used to clone c-K(B)-Ras from G12V-K(B)-Ras/pZip (gift from J. Gibbs, Merck) where the forward N-terminal primer was extended beyond the 12th codon to back-mutate the valine 12 to the wild-type glycine (5′-ACACCATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTA-3′). The reverse complement or the C-terminal region of c-K(B)-Ras was used for the reverse primer (3′-AGATCTCCATGGGTCGACTATTTACATAATTACACACTTTG-5′). The resulting c-K(B)-Ras/pTargeT was transfected into N-Ras knockout cells as described. Prior to transfections, the c-K(A)-and c-K(B)-Ras plasmids were sequenced to confirm their identity with the sequences of mouse c-K(A)- or c-K(B)-Ras in the GenBank data base. All transfected clones were tested for the presence and level of expression of the transgene by Western analysis. All lysis buffers contained the following phosphatase inhibitors: 30 mm β-glycerophosphate, 5 mm p-nitrophenyl phosphate, 1 mm each of phosphoserine and phosphothreonine, 0.2 mm phosphotyrosine, 100 μm sodium vanadate, and the following protease inhibitors: 50 μg/ml each of aprotinin and leupeptin, 25 μg/ml pepstatin A, and 1 mm phenylmethanesulfonyl fluoride. For Western analysis of Ras expression, serine 473-phospho-Akt (pAkt) levels, total Akt levels, and phospho-MAP kinase (pMAPK levels), cells were harvested by scraping into PBS, and the resulting cell pellet was resuspended in p21 buffer (20 mm MOPS, 5 mmMgCl2, 0.1 mm EDTA, 200 mm sucrose, pH 7.4) containing 1% CHAPS (U.S. Biochemical Corp.) and incubated for 20 min on ice. The lysate was centrifuged again at 13,000 ×g, and the supernatant was retained. Protein concentration was determined by the method of Bradford (50.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). For Western analysis of total Bad or phospho-Bad levels, cells were harvested by trypsinization, combined with their medium, and centrifuged at 1000 × g for 10 min. The cells were washed once in Tris-buffered saline (TBS; 20 mm Tris, 140 mmNaCl, pH 7.4) and solubilized in TBS containing 1% Nonidet P-40 (Igepal, Sigma) and phosphatase and protease inhibitors as described. After 20 min on ice, the lysate was centrifuged at 13,000 ×g, and the supernatant was retained for protein measurements and Western analysis. Lysates containing equal amounts of protein were loaded onto SDS-polyacrylamide gels. Following electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) (Hybond P; Amersham Pharmacia Biotech). Blocking was performed in 5% nonfat milk containing 5% newborn calf serum (Life Technologies). Blots were incubated with primary antibodies for 2–3 h at room temperature or overnight at 4 °C followed by washing in TBS, 0.1% Tween. The blots were incubated with either goat anti-mouse horseradish peroxidase (HRP) (Kirkegaard and Perry Laboratories) or anti-rabbit HRP (Transduction Laboratories). After washing, the blots were developed, as indicated, with ECL (Amersham Pharmacia Biotech) and exposure to film (Hyperfilm ECL; Amersham Pharmacia Biotech) or with ECL-Plus (Amersham Pharmacia Biotech) and detection with a Molecular Dynamics Storm Imager. Untreated cells or cells treated for the indicated times were harvested by trypsinization and combined with their medium (to collect any detached cells), centrifuged, and washed once in cold PBS. The cell pellets were resuspended in 1% paraformaldehyde (EM Science) in PBS and incubated on ice for 15 min. The fixed cells were centrifuged and washed once with PBS and resuspended in cold 70% ethanol. TUNEL analysis was performed by fluorescence-activated cell sorting using the APO-BRDU flow cytometry kit for apoptosis according to the manufacturer's directions (Phoenix Flow; Pharmingen). Untreated or treated cells in 12-well cluster plates were scraped in their medium and centrifuged at 500 × 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). 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, according to the manufacturer's instructions. Incubation was performed overnight at 4 °C instead of 2–3 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. Expression of c-N-Ras is absent in all immortalized N-Ras knockout cell lines (N−/−) (Fig.1, top). The expression levels of c-N-Ras in the N-Ras knockout cells ectopically expressing c-N-Ras (N−/−wtN) are similar to that observed in the control N+/+ cells (Fig. 1, bottom). All cell lines, except K(i)-Ras knockout cells, express K(i)-Ras (see below), and none express detectable levels of Ha-Ras (data not shown). Since the N-Ras knockout cells express only c-K(A)- and c-K(B)-Ras, they present a unique system to examine signaling systems that might specifically require c-N-Ras. We chose to test for changes in either phospho-MAP kinase or phospho-Akt levels, since each of these is regulated through a distinct Ras signaling pathway (Raf-1 and PI 3-kinase, respectively). Differences between N-Ras knockout cells and control cells in the level of activated MAP kinase or Akt were examined both at steady state and following agonist stimulation. We examined phospho-MAP kinase (p42 and p44) levels under steady-state growth and following treatment with TNFα in the presence of cycloheximide (Fig.2 A). The N-Ras knockout cells, control N+/+ cells, and N-Ras knockout cells stably expressing c-N-Ras at control levels possessed similar levels of phosphorylated MAP kinase at steady state and following treatment with TNFα. There was a small increase in the level of phospho-MAP kinase at 1 h that decreased to steady-state levels after 4 h. This is consistent with the report that both Jun N-terminal kinases and extracellular signal-related kinases (ERKs) are activated in a Ras-dependent manner following Fas ligation in SHEP cells (51.Goillot E. Raingeaud J. Ranger A. Tepper R.I. Davis R.J. Harlow E. Sanchez I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3302-3307Crossref PubMed Scopus (246) Google Scholar). Recently, two groups reported that phosphorylation of Bad on serine 112 is regulated by the MAP kinase pathway (31.Scheid M.P. Schubert K.M. Duronio V. J. Biol. Chem. 1999; 274: 31108-31113Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 52.Fang X., Yu, S. Eder A. Mao M. Bast Jr., R.C. Boyd D. Mills G.B. Oncogene. 1999; 18: 6635-6640Crossref PubMed Scopus (233) Google Scholar). The results from these studies suggested that the MAP kinase pathway is necessary for Ser112 phosphorylation and inactivation of proapoptotic Bad, similar to Ser136 phosphorylation of Bad by Akt (32.Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4895) Google Scholar, 33.del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1974) Google Scholar, 34.Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2233) Google Scholar, 53.Blume-Jensen P. Janknecht R. Hunter T. Curr. 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Our results demonstrate that, at steady state, the N-Ras knockout cells possess minimal levels of pAkt in contrast to control cells (Fig. 2 B,upper panel). Ectopic expression of c-N-Ras in the N-Ras knockout cells significantly restores the level of pAkt to levels comparable with those observed in the control cells. The differences observed in pAkt are not a result of differences in the total amount of Akt (Fig. 2 B, bottom panel). The N-Ras knockout cells, control N+/+ cells, and the N-Ras knockout cells ectopically expressing c-N-Ras (N−/−wtN) possess similar levels of total Akt protein both at steady state and following treatment with TNFα. This implies that activation of the c-N-Ras/PI 3-kinase/Akt pathway may be impaired in N-Ras knockout cells. Bad can be phosphorylated on position 136 by Akt (32.Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4895) Google Scholar, 33.del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1974) Google Scholar, 34.Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Tex
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