c-Fos Degradation by the Proteasome
1998; Elsevier BV; Volume: 273; Issue: 39 Linguagem: Inglês
10.1074/jbc.273.39.25015
ISSN1083-351X
AutoresHuiling He, Xiao-Mei Qi, Johannes Grossmann, Clark Distelhorst,
Tópico(s)Cell death mechanisms and regulation
Resumoc-Fos is a transcription factor that promotes cell growth, differentiation, and transformation. We found that c-Fos was degraded when WEHI7.2 mouse lymphoma cells were induced to undergo apoptosis with the calcium ATPase inhibitor, thapsigargin, or the glucocorticoid hormone, dexamethasone. The degradation of c-Fos preceded caspase-3 activation and apoptotic nuclear chromatin condensation and was inhibited by the proteasome inhibitors MG132,N-acetyl-leucyl-leucyl-norleucinal, and lactacystin. Stable transfection of WEHI7.2 cells with a mutant form of c-Fos that was not degraded by the proteasome inhibited apoptosis. Also, overexpression of Bcl-2 in WEHI7.2 cells blocked c-Fos degradation and inhibited apoptosis. The results indicate that proteasome-mediated degradation of c-Fos is an early, Bcl-2-regulated step in apoptosis induction by thapsigargin and dexamethasone. These findings suggest that c-Fos may have a protective action that is eliminated by proteasome-mediated degradation and preserved by Bcl-2. c-Fos is a transcription factor that promotes cell growth, differentiation, and transformation. We found that c-Fos was degraded when WEHI7.2 mouse lymphoma cells were induced to undergo apoptosis with the calcium ATPase inhibitor, thapsigargin, or the glucocorticoid hormone, dexamethasone. The degradation of c-Fos preceded caspase-3 activation and apoptotic nuclear chromatin condensation and was inhibited by the proteasome inhibitors MG132,N-acetyl-leucyl-leucyl-norleucinal, and lactacystin. Stable transfection of WEHI7.2 cells with a mutant form of c-Fos that was not degraded by the proteasome inhibited apoptosis. Also, overexpression of Bcl-2 in WEHI7.2 cells blocked c-Fos degradation and inhibited apoptosis. The results indicate that proteasome-mediated degradation of c-Fos is an early, Bcl-2-regulated step in apoptosis induction by thapsigargin and dexamethasone. These findings suggest that c-Fos may have a protective action that is eliminated by proteasome-mediated degradation and preserved by Bcl-2. thapsigargin dexamethasone N-acetyl-leucyl-leucyl-norleucinal N-acetyl-leucyl-leucyl-methional 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid N-acetyl-Asp-Glu-Val-Asp-7-amino- 4-methylcoumarin. Apoptosis, or programmed cell death, is a genetically regulated process necessary for maintenance of normal tissue homeostasis (1Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2425) Google Scholar). In the nematode, Caenorhabditis elegans, apoptosis is regulated by the death effector gene, ced-3, and the death inhibitor gene, ced-9 (2Hengartner M.O. Ellis R.E. Horvitz H.R. Nature. 1992; 356: 494-499Crossref PubMed Scopus (712) Google Scholar, 3Yuan J. Shaham S. 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We have investigated the involvement of c-Fos in apoptosis in the murine cell line WEHI7.2, a tissue culture model of glucocorticoid-induced apoptosis in immature thymocytes (31Flomerfelt R.A. Miesfeld R.L. J. Cell Biol. 1994; 127: 1729-1742Crossref PubMed Scopus (25) Google Scholar). WEHI7.2 cells do not express detectable levels of Bcl-2 and undergo apoptosis in response to thapsigargin (TG),1 a selective inhibitor of the endoplasmic reticulum-associated calcium-ATPase, and dexamethasone (DX), a synthetic glucocorticosteroid hormone (32Lam M. Dubyak G. Chen L. Nuñez G. Miesfeld R.L. Distelhorst C.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6569-6573Crossref PubMed Scopus (609) Google Scholar). Here we report that (i) both TG and DX induce rapid degradation of c-Fos by the proteasome, (ii) proteasome-mediated c-Fos degradation precedes caspase activation and apoptotic nuclear chromatin condensation, (iii) Bcl-2 overexpression inhibits c-Fos degradation by the proteasome and increases the level of c-Fos expression, and (iv) a C-terminal truncated version of c-Fos that evades degradation by the proteasome inhibits apoptosis induction by TG and DX. The anti-c-Fos antibody was purchased from Upstate Biotechnology Inc. The anti-CPP32 p17 subunit antibody (SK398) was raised to amino acids 85–94 of caspase-3 (VRNKNDLTRE) and provided by Kristy Kikly (SmithKline Beecham Pharmaceuticals). MG132 (Z-Leu-Leu-Leu-H aldehyde) was purchased from Peptides International, Inc. Lactacystin was purchased from E. J. Corey (Harvard University). N-Acetyl-leucyl-leucyl-norleucinal (ALLN) and N-acetyl-leucyl-leucyl-methional (ALLM) were purchased from Boehringer Mannheim. Benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone was purchased from Enzyme Systems Products. E64d ((2s,3s)-trans-epoxy succinyl-l-leucylamido-3-methylL-butaneethyl-estero) was purchased from Sigma. Stock solutions of these reagents were prepared in dimethyl sulfoxide and stored in aliquots at −80 °C, except for lactacystin, which was dissolved in water. DX was purchased from Sigma, and TG was purchased from LC Laboratories. Cells were cultured in Dulbecco's modified Eagle's medium (BioWhittaker) supplemented with 2 mm glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin, 0.4 mm nonessential amino acids, and 10% (v/v) heat-inactivated horse serum (Hyclone) at 37 °C in a 7% CO2 atmosphere. l-Glutamine, antibiotics, and nonessential amino acids were from Life Technologies, Inc. TG was added to cells from a stock solution in Me2SO to give a final concentration of 100 nm. DX was added to cells from a stock solution in ethanol to give a final concentration of 1 μm. Viable cells, defined as cells that exclude trypan blue dye, were counted using a hemocytometer. The percentage of cells with an apoptotic nuclear morphology was assessed by fluorescence microscopy after staining with eithidium bromide and acridine orange (33Distelhorst C.W. McCormick T.S. Cell Calcium. 1996; 19: 473-483Crossref PubMed Scopus (52) Google Scholar). The c-fos cDNA was removed from the pEMSV-fos plasmid (provided by Michael Simonson, Case Western Reserve University) by digestion withEcoRI and ligated in sense orientation into the pcDNA-3 expression vector (Invitrogen) downstream of the cytomegalovirus promotor, producing a pcDNA-3-sense-fos vector. The orientation of inserts was confirmed by analysis with four groups of restriction enzymes. The c-fosΔC cDNA was cloned by polymerase chain reaction using the 5′ primer sequence AAAGGGGAATTCATGTACCCATACGACGTCCCAGACTACGCTATGTTCTCGGGTTTCAACGCGGAC and the 3′ primer sequence ACAGTGGAATTCTTATTATTTCGGTGGGCAGCTGCGCAGCTA. The c-fosΔC cDNA was cloned into the pcDNA-3 expression vector in the sense orientation, confirmed by DNA sequencing. The empty pcDNA-3 vector and the pcDNA-3-fosΔC vectors were transfected into WEHI7.2 cells by electroporation and selected with G418 (1 mg/ml). Findings were confirmed in experiments in which the c-fos cDNA was cloned in sense orientation in the pSFFV-neo expression vector. To recover c-Fos, cells were washed twice with cold phosphate-buffered saline, resuspended in a 5-fold volume of modified RIPA buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 1 mmEDTA, 5 mm EGTA, 1 mm NaVO4, 5 mm NaF, 20 mm N-ethylmaleimide, 1 mm phenylmethanesulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin), and boiled immediately for 10 min. To recover caspase-3, cells were lysed in a buffer containing 10 mmHEPES/KOH, pH 7.4, 2 mm EDTA, 0.1% CHAPS, 5 mmdithiothreitol, 1 mm phenylmethanesulfonyl fluoride, 10 mg/ml pepstatin A, 1 mg/ml leupeptin, and 1 mg/ml aprotinin. The cell lysates were centrifuged at 14,000 × g for 10 min. The protein concentration of supernatant samples was determined by the Bio-Rad protein assay (Bio-Rad Laboratories). 70 μg of each protein lysate was separated by SDS-polyacrylamide gel electrophoresis under reducing and denaturing conditions and transferred to polyvinylidene difluoride membrane (Millipore Co.). Equal loading of protein lysates was confirmed by staining membranes with Ponseau S or staining gels with Coomassie Blue after protein transfer. Membranes were incubated in 5% nonfat dry milk in T-TBS (18 mm Tris-HCl, pH 7.6, 122 mm NaCl, 0.1% Tween 20) at room temperature for 2 h and then incubated with the primary antibodies (anti-c-Fos at 1:5000 dilution or anti-caspase-3 at 1:4000 dilution) at 4 °C overnight. Membranes were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (Life Technologies, Inc.) at a 1:2000 dilution. Immune complexes were detected with the ECL Western blotting detection reagent (Amersham Pharmacia Biotech) according to the manufacturer's protocol, followed by exposure to x-ray film (Sigma). Cells were lysed in 0.25 ml of lysis buffer (100 mm HEPES, 10% sucrose, 0.1% CHAPS, 1 mm phenylmethanesulfonyl fluoride, 10 mmdithiothreitol (DEVD-AMC fluorogenic assay only), 100 μmpepstatin, 100 μm leupeptin, 1 mm EDTA at pH 7.5), followed by a 30-min incubation on ice. Lysis was completed by two 10-s sonication pulses (Sonicator XL, Heat Systems, Farmingdale, NY). Cellular debris was removed by centrifugation, and the cytosolic extract was stored at −20 °C. Protein content of the lysates was determined by the Bio-Rad Protein assay according to the manufacturer's instructions. 20 μg of cytosolic protein and 50 μm DEVD-AMC (Biomol Research Laboratory, Plymouth Meeting, PA) were incubated in a total volume of 500 μl in 100 mm HEPES, 10% sucrose, 0.1% CHAPS, pH 6.8, for 30 min on an orbital shaker at 37 °C. Samples were diluted with lysis buffer to a final volume of 1 ml immediately before measurement of fluorescence by fluorospectrophotometry (Perkin-Elmer LS-3; excitation, 380 nm; emission, 480 nm; slit width, 5.0 nm). Standards containing 0–5000 pmol of AMC were used to determine the amount of fluorochrome released. Measurements were recorded over the linear range of the assay. Bovine serum albumin (50 μg) served as negative control. Cells were pretreated with inhibitors for time periods described in the text at the following concentrations: MG132, 25 μm; lactacystin, 10 μm; ALLN, 50 μm; ALLM, 50 μm; benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone, 50 μm; E64d, 10 μm. The level of c-Fos was measured by Western blotting in wild type WEHI7.2 cells, which lack Bcl-2, and in stable transfectants, W.Hb12, which were derived from WEHI7.2 cells and express a high level of Bcl-2 (32Lam M. Dubyak G. Chen L. Nuñez G. Miesfeld R.L. Distelhorst C.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6569-6573Crossref PubMed Scopus (609) Google Scholar). Findings were confirmed in replicate experiments using vector only control transfectants, WEHI7.2-neo, and another clone that stably expresses a high level of Bcl-2, W.Hb15 (not shown). The basal level of c-Fos, before treatment with either TG or DX, was 4.6 ± 2.1-fold (mean ± S.E.) higher in W.Hb12 cells than in WEHI7.2 cells (Fig. 1 A). After adding 100 nm TG to WEHI7.2 cells, the level of 66-kDa c-Fos declined markedly, giving rise to a 46-kDa fragment (Fig. 1 A), which in many experiments was further degraded to smaller size fragments. In contrast, the level of c-Fos was stable following TG treatment in W.Hb12 cells (Fig. 1 A). A decline in c-Fos level was also induced when WEHI7.2 cells were treated with 1 μm DX, although the appearance of the 46-kDa fragment was more variable than in TG-treated cells, perhaps due to the longer time course of DX-induced cell death (Fig. 2 A). In contrast, c-Fos was stable when W.Hb12 cells were treated with DX (Fig. 2 A). In the case of both TG- and DX-treated WEHI7.2 cells, the decline in c-Fos preceded caspase-3 (CPP32, apopain, and Yama) activation, detected both by generation of a p17 caspase-3 activation fragment (Figs. 1 B and 2 B) and by cleavage of a synthetic tetrapeptide substrate, DEVD-AMC (Figs. 1 C and 2 C). Furthermore, the decline in c-Fos preceded apoptotic nuclear chromatin condensation detected by fluorescence microscopy (Figs. 1 D and 2 D). Consistent with its known antiapoptotic action, Bcl-2 inhibited both caspase-3 activation (Figs. 1, B and C, and 2, B and C) and nuclear chromatin condensation (Figs. 1 D and 2 D). Thus, both TG and DX induced a marked decline in the level of c-Fos, detected during the early phase of apoptosis and inhibited by Bcl-2.Figure 2Degradation of c-Fos in DX-treated cells. A, Western blot of c-Fos in protein extracts from DX-treated cells. Whole cell extracts were prepared from WEHI7.2 cells (− Bcl-2) and W.Hb12 cells (+ Bcl-2) at time intervals after adding 1 μm DX and Western blot analysis was done with anti-c-Fos antibody. B, Western blot of caspase-3 activation fragment in protein extracts from cells treated with DX. Whole cell extracts were prepared from WEHI7.2 cells (− Bcl-2) and W.Hb12 cells (+ Bcl-2) at time intervals after adding 1 μm DX and Western blot analysis was done with an antibody that recognizes the 17-kDa activation fragments of caspase-3 but not the intact proenzyme. C, fluorogenic substrate assay measuring caspase-3-mediated cleavage of the synthetic peptide substrate DEVD-AMC. Protein extracts were prepared from WEHI7.2 cells (− Bcl-2) and W.Hb12 cells (+ Bcl-2) at time intervals after adding 1 μm DX. The data represent the means ± S.E. of three separate experiments. D, summary of morphological assays for apoptosis in DX-treated cells. WEHI7.2 cells (− Bcl-2) and W.Hb12 cells (+ Bcl-2) were stained with acridine orange and ethidium bromide at time intervals after adding 1 μm DX. The percentage of cells displaying apoptotic nuclear chromatin condensation is shown as the mean ± S.E. of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm that the decline in c-Fos induced by TG was due to accelerated degradation and to identify the responsible protease, WEHI7.2 cells were treated with protease inhibitors before adding TG (Fig. 3 A). The decline in c-Fos and the appearance of the 46-kDa c-Fos fragment were inhibited by pretreating cells with the specific proteasome inhibitors, lactacystin and MG132 (N-carbobenzoxyl-l-leucinyl-l-leucinyl) (34Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1915) Google Scholar, 35Fenteany G. Standaert R.F. Lane W.S. Chois S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1496) Google Scholar) and also by the less specific proteasome inhibitor, ALLN (36Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2186) Google Scholar). ALLN also exhibits significant inhibitory activity for the cysteine protease calpain (36Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2186) Google Scholar). Although c-Fos is subject to degradation by calpain (37Carillo S. Pariat M. Steff A.-M. Roux P. Etienne-Julan M. Lorca T. Piechaczyk M. Oncogene. 1989; 9: 1079-1089Google Scholar), it is unlikely that calpain played a significant role, because c-Fos degradation was not prevented by the calpain inhibitors, ALLM and E64d (36Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2186) Google Scholar) (Fig. 3 A). Pretreatment with benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone, a cell permeant inhibitor of the caspase family of cysteine proteases (38Zhu H. Fearnhead H.O. Cohen G.M. FEBS Lett. 1995; 374: 303-308Crossref PubMed Scopus (146) Google Scholar), did not inhibit c-Fos degradation (Fig. 3 A), indicating that proteasome-mediated degradation of c-Fos is not secondary to caspase activation. The decline in c-Fos observed in DX-treated WEHI7.2 cells was also due to proteasome-mediated degradation, based on evidence that c-Fos was stabilized by pretreatment with the proteasome inhibitors lactacystin, MG132, and ALLN (Fig. 3 B) and not by E64d (not shown). In DX-treated WEHI7.2 cells, pretreatment with proteasome inhibitors stabilized 46–48 kDa degradation intermediates as well as intact c-Fos (Fig. 3 B). Also, several higher molecular mass forms were detected in DX-treated cells in the presence of proteasome inhibitors (Fig. 3 B). The appearance of both degradation intermediates and presumably polyubiquitinated high molecular weight forms in the presence of proteasome inhibitors has been reported previously in the case of other proteasome target proteins (39Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1129) Google Scholar, 40Wiertz E.J.H.J. Jones T.R. Sun L. Bogyo M. Geuze H.J. Ploegh H.L. Cell. 1996; 84: 769-779Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar). In view of the preceding findings suggesting a correlation between proteasome-mediated c-Fos degradation and apoptosis in WEHI7.2 cells, we wished to test whether c-Fos degradation by the proteasome contributes to apoptosis induction. It was not possible to answer this question with the use of proteasome inhibitors, because treatment of WEHI7.2 cells with lactacystin and MG132 induces apoptosis. 2H. He and C. W. Distelhorst, unpublished data. Therefore, as an alternative approach, WEHI7.2 cells were stably transfected with either a cDNA encoding wild type c-Fos or a cDNA encoding a truncated version of c-Fos, c-FosΔC, missing 21 C-terminal amino acids. This strategy was based on evidence that deletion of the C-terminal sequence stabilizes c-Fos by reducing proteasome-mediated degradation (23Tsurumi C. Ishida N. Tamura T. Kakizuka A. Nishida E. Okumura E. Kishimoto T. Inagaki M. Okazaki K. Sagata N. Ichihara A. Tanaka K. Mol. Cell. Biol. 1995; 15: 5682-5687Crossref PubMed Scopus (127) Google Scholar). Wild type c-Fos was still degraded following TG treatment, even when expressed at levels 2–4-fold higher than the endogenous level (Fig. 4 A), and therefore did not provide protection against apoptosis (Fig. 4 C). In contrast, c-FosΔC was stable for 16–20 h after TG treatment (Fig. 4 A) or DX treatment (Fig. 5 A). In both situations, c-FosΔC expression inhibited the induction of both caspase-3 activation (Figs. 4 B and 5 B) and apoptotic nuclear chromatin condensation (Fig. 4 C and 5 C). The protective effect of c-FosΔC is not associated with alterations in the level of expression of endogenous Bcl-2 (data not shown). In summary, a mutant form of c-Fos that evades degradation by the proteasome inhibits apoptosis induction by both TG and DX. Thus, it appears that inhibiting the degradation of c-Fos prevents apoptosis, suggesting that accelerated degradation of c-Fos contributes to the generation of apoptotic cell death in WEHI7.2 cells.Figure 5Inhibition of DX-induced apoptosis by a stable mutant of c-Fos. A, Western blot of the C-terminal truncation mutant of c-Fos, c-FosΔC, in protein extracts from DX-treated cells. WEHI7.2 cells were transfected with either control empty vector or a vector encoding c-FosΔC, and stable transfectants were selected for resistance to G418. Whole cell extracts were prepared at time intervals after adding 1 μm DX. Western blot analysis was done with anti-c-Fos antibody. B, Western blot analysis of caspase-3 activation fragment in protein extracts from WEHI7.2 cells stably transfected with vector encoding either neo or c-FosΔC. Cells were treated with 1 μm DX. Western blot analysis was done with an antibody that recognizes the 17-kDa activation fragment of caspase-3 but not the intact proenzyme.C, summary of morphological assays for apoptosis in DX-treated cells. Control neo transfectants and c-FosΔC transfectants were stained with acridine orange and ethidium bromide at time intervals after after adding 1 μm DX. The percentage of cells displaying apoptotic nuclear chromatin condensation is shown as the mean ± S.E. of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our findings indicate for the first time that c-Fos degradation by the proteasome contributes to the execution of the apoptotic death program. It appears that c-Fos may confer protection against TG- and DX-induced apoptosis that is abrogated by proteasome-mediated degradation and preserved either by expressing a C-terminal truncated version of c-Fos that is not degraded by the proteasome or by overexpressing Bcl-2, which inhibits c-Fos degradation by the proteasome. The concept that c-Fos has an antiapoptotic action is consistent with other recent evidence that c-Fos mediates expression of genes that protect against apoptosis (41Schreiber M. Baumann B. Cotten M. Angel P. Wagner E.F. EMBO J. 1995; 14: 5338-5349Crossref PubMed Scopus (178) Google Scholar, 42Kaina B. Haas S. Kappes H. Cancer Res. 1997; 57: 2721-2731PubMed Google Scholar, 43Ivanov V.N. Nikolic-Zugic J. J. Biol. Chem. 1997; 272: 8558-8566Abstract Full Text Full Text PDF PubMed Google Scholar) and with evidence that cells from c-fos −/− knockout mice are hypersensitive to apoptosis induction (42Kaina B. Haas S. Kappes H. Cancer Res. 1997; 57: 2721-2731PubMed Google Scholar). Earlier reports suggested that c-fos may be a mediator of apoptosis, a concept based on evidence that c-fostranscription increases in cells undergoing apoptosis (44Buttyan R. Zakeri Z. Lockshin R. Wolgemuth D. Mol. Endocrinol. 1988; 2: 650-657Crossref PubMed Scopus (322) Google Scholar, 45Colotta F. Polentarutti N. Sironi M. Mantovani A. J. Biol. Chem. 1992; 267: 18278-18283Abstract Full Text PDF PubMed Google Scholar, 46Smeyne R.J. Vendrell M. Hayward M. Baker S.J. Miao G.G. Schilling K. Robertson L.M. Curran T. Morgan J.I. 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We thank Michael Simonson for antibodies, plasmids, and helpful suggestions, Kristy Kikly for antibodies to caspase-3, and Jack Hensold for lactacystin and antibodies to ubiquitin. We also thank Mark Distelhorst for preparing figures.
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