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Translational Control of the Antiapoptotic Function of Ras

Translational Control of the Antiapoptotic Function of Ras THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 32, Issue of August 11, pp. 24776 –24780, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Received for publication, March 7, 2000, and in revised form, April 28, 2000 Published, JBC Papers in Press, May 12, 2000, DOI 10.1074/jbc.M001938200 Vitaly A. Polunovsky‡, Anne-Claude Gingras§, Nahum Sonenberg§, Mark Peterson‡, Annie Tan‡, Jeffrey B. Rubins‡, J. Carlos Manivel¶, and Peter B. Bitterman‡i From the ‡Department of Medicine and the ¶Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota 55455 and the §Department of Biochemistry, McGill University, Montreal, Quebec H3G IY6, Canada Activated Ras has been shown to provide powerful the intrinsic apoptotic machinery. Akt also regulates the FK506 binding-protein 12 (FKBP12)-rapamycin-associated antiapoptotic signals to cells through well defined tran- scriptional and post- translational pathways, whereas protein/mammalian target of rapamycin (FRAP/mTOR), a ki- translational control as a mechanism of Ras survival nase which functions in the control of translation by activating signaling remains unexplored. Here we show a direct two components of the protein synthesis apparatus: (i) the relationship between assembly of the cap-dependent initiation complex binding the 59-mRNA cap and (ii) the 40 S s6k translation initiation apparatus and suppression of apo- ribosomal protein S6 kinase, p70 (8 –11). However, data ptosis by oncogenic Ras in vitro and in vivo. Decreasing examining the importance of translational control in the mech- protein synthesis with rapamycin, which is known to anism of Ras survival signaling are lacking. inhibit cap-dependent translation, increases the suscep- Translational control is usually exerted at the initiation step. tibility of Ras-transformed fibroblasts to cytostatic In eukaryotes, the 59-mRNA cap is bound by the eukaryotic drug-induced apoptosis. In contrast, suppressing global translation initiation complex eIF4F, which consists of a bi- protein synthesis with equipotent concentrations of cy- directional RNA helicase eIF4A, the docking protein eIF4G and cloheximide actually prevents apoptosis. Enforced ex- the cap binding subunit eIF4E (12). A major target for regula- pression of the cap-dependent translational repressor, tion, eIF4E is considered to be rate limiting for translation the eukaryotic translation initiation factor (eIF) 4E- initiation under most circumstances (13), and its up-regulation binding protein (4E-BPI), sensitizes fibroblasts to apo- is associated with cell proliferation, suppression of apoptosis, ptosis in a manner strictly dependent on its ability to and tumorigenicity (14, 15). The function of eIF4E is inhibited sequester eIF4E from a translationally active complex by members of the family of translational repressors, the with eIF4GI and the co-expression of oncogenic Ras. eIF4E-binding proteins (4E-BPs, also known as PHAS) (13, 16). Ectopic expression of 4E-BP1 also promotes apoptosis of When hypophosphorylated, 4E-BPs compete with eIF4G for Ras-transformed cells injected into immunodeficient binding to eIF4E and sequester eIF4E in a nonfunctional com- mice and markedly diminishes their tumorigenicity. These results establish that eIF4E-dependent protein plex. Upon hyperphosphorylation, 4E-BPs dissociate from the synthesis is essential for survival of fibroblasts bearing complex with eIF4E allowing it to form an active translation oncogenic Ras and support the concept that activation initiation complex (16, 17). To elucidate the role of transla- of cap-dependent translation by extracellular ligands or tional control in Ras survival signaling, here we focus directly intrinsic survival signaling molecules suppresses apo- on the 59-mRNA cap binding complex and examine the induc- ptosis, whereas synthesis of proteins mediating apo- tion of apoptosis in cells transformed with oncogenic Ras after ptosis can occur independently of the cap. a generalized reduction in protein synthesis or after specific repression of cap-dependent translation initiation. EXPERIMENTAL PROCEDURES Extracellular survival factors suppress the intrinsic apo- Generation of Clones and Transient Transfection—Cloned rat em- ptotic apparatus through cognate receptor kinases at the cell bryo fibroblasts (CREF) and CREF/RasV12 (a gift from A. De Benedetti) surface, which activate the proto-oncogene ras and a number of were subcloned and maintained in Dulbecco’s modified Eagle’s medium pleiotropic transcriptional and post-translational effector path- supplemented with 10% fetal calf serum. The coding sequence of human ways (1). A major effector of Ras survival signaling is the 4E-BP1 was amplified by polymerase chain reaction and directionally serine/threonine protein kinase, Akt (2, 3). Transcriptional con- cloned into the EcoRI and BamHI sites of the mammalian expression trol is exerted by Akt-mediated phosphorylation of the Fork- vector pSRapuro, (a kind gift from Dr. P. Jolicoeur, Institut de Recher- ches Cliniques, Montreal). To generate the eIF4E binding site deletion head1 family transcription factor FKHRL1(4) and the tran- mutant (amino acids 54 – 63; 4E-BP1-D), the 4E-BP1 coding sequence scription factor nuclear factor-kB (5), which alter expression of was cloned into the cytomegalovirus-based vector pACTAG-2, which apoptosis-related genes. Akt-mediated phosphorylation of the was used as a template for polymerase chain reaction site-directed Bcl-2 family member Bad (6) and the cell death protease mutagenesis (18). Clones of CREF and CREF/RasV12 expressing wild caspase-9 (7) is implicated in post-translational suppression of type or mutant 4E-BP1 were generated using the FuGENE 6 (Roche Diagnostics) transfection technique. Selection of transfected cells was begun after 24 h with medium containing 1 mg/ml puromycin, and * This work was supported by NHLBI, National Institutes of Health- resistant clones were isolated after 12–16 days. CREF/RasV12s were funded SCOR Grant 2P50-HL50152, a grant from the National Cancer also transiently transfected with a pACTAG-2 construct encoding he- Institute of Canada, a doctoral award from the Medical Research Coun- magglutinin (HA)-tagged human 4E-BP1-wt, 4E-BP1-D, or a vector cil of Canada, and the M.D. Ph.D. program of the University of Minne- carrying only the HA tag. To detect the level of HA expression by flow sota Medical School. The costs of publication of this article were de- frayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 The abbreviations used are: FRAP/mTOR, FKBP12-rapamycin-as- U.S.C. Section 1734 solely to indicate this fact. sociated protein/mammalian target of rapamycin; eIF, lukaryotic trans- To whom correspondence should be addressed. Tel.: 612-624-0999; lation initiation factor; 4E-BP, eIF4E-binding protein; CREF, cloned rat Fax: 612-625-2174; E-mail: [email protected].. embryo fibroblasts; HA, hemagglutinin; BP1-wt, wild type 4E-BP1. 24776 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Translational Repressor 4E-BP1 Inhibits Ras Survival Signaling 24777 FIG.1. Rapamycin suppresses Ras- induced chemoresistance. a, expres- sion of Ras protein in CREF/Neo and CREF/RasV12. b, apoptosis was assessed after incubation (24 h, 37 °C) with or without 75 nM rapamycin in medium alone (solid bar) or medium supple- mented either with 5 mM lovastatin (striped bar)or75nM camptothecin (open bar). Values shown represent the mean 6 S.D. (n 5 3) of the percentage of cells with subdiploid DNA content. c and d, lovasta- tin-induced apoptosis in association with suppression of protein synthesis by ra- pamycin or cycloheximide. The rates of protein synthesis and lovastatin-induced apoptosis were quantified in parallel cul- tures exposed for 24 h to different con- centrations of either rapamycin (c)or cycloheximide (d). Protein synthesis is displayed as a percent of that observed without inhibitors. cytometry, cells were fixed with absolute ethanol and incubated for 16 h expressed oncogenic RasV12 enables CREF to survive in oth- at 4 °C with mouse anti-HA IgG2 antibody (4 mg/ml, Roche Molecular bk erwise lethal concentrations of cytostatic drugs (nongenotoxic, Biochemicals) or with mouse isotype-specific IgG2 antibody (4 mg/ml, bk lovastatin; genotoxic, camptothecin, Fig. 1). The FRAP/mTOR PharMingen) followed by incubation with fluorescein-conjugated anti- inhibitor rapamycin completely abrogated Ras-dependent re- mouse IgG antibody (1:40, Sigma) for 30 min. Immunoblot Analysis of Cap-bound Proteins—Cell lysates (250 ml sistance to drug-induced cell death (Fig. 1b) and even when containing 250 mg of protein) were incubated with m GTP-Sepharose applied as a single agent, stimulated apoptosis in cells express- resin (Amersham Pharmacia Biotech) to capture eIF4E and its binding ing activated Ras. This proapoptotic effect of rapamycin was partners (9). Samples were eluted with buffer containing 70 mM m GTP. not observed in nontransformed fibroblasts. These observa- Cap-bound material was subjected to SDS-polyacrylamide gel electro- tions confirm a dual proapoptotic and antiapoptotic function for phoresis and transferred to nitrocellulose. Blots were probed first for RasV12 (2) and implicate FRAP/mTOR in Ras-dependent res- eIF4E (mouse monoclonal antibody, 1:500, Transduction Laboratories), then stripped and probed for 4E-BP1 (rabbit polyclonal antiserum cue from both Ras-activated and drug-triggered apoptotic 1:2500) (19), and stripped and probed a third time for eIF4GI (rabbit pathways. polyclonal antibody 1:4000) (20) When rapamycin was added to Ras-transformed cells, it Apoptosis Assays—Frequency of apoptosis was quantified by flow caused a dose-dependent decline in protein synthesis, which cytometric analysis of the percentage of cells with hypodiploid DNA paralleled its ability to sensitize cells to lovastatin-induced content. Adherent and nonadherent cells were pooled, washed in phos- phate-buffered saline, and fixed with ice-cold 70% ethanol for at least apoptosis (Fig. 1c). Of note, equipotent doses of the peptide 1 h. Fixed cells were washed and incubated in propidium iodide stain elongation inhibitor cycloheximide actually blocked apoptosis mixture 50 mg/ml propidium iodide, 0.05% Triton X-100, 37 mg/ml (Fig. 1d), demonstrating that the execution of lovastatin-in- EDTA, 100 units/ml ribonuclease in phosphate-buffered saline). After duced cell death requires global protein synthesis. These re- incubation for 45 min at 37 °C, DNA content was determined by quan- sults suggest that a generalized inhibition of mRNA transla- titative flow cytometry using standard optics of the FACScan flow cytometer (Becton Dickinson) and the CellQuest program. tion is not the means by which rapamycin exerts its Tumorigenicity Assay—Under sterile conditions, 3 3 10 cells in proapoptotic effect, rather they point toward a selective inhi- phosphate-buffered saline were injected into each flank of immunodefi- bition of antiapoptotic mRNA translation or a mechanism in- cient mice (Nu Nu, Harlan). CREF/RasV12 cells were injected into one dependent of its ability to repress translation. flank of each pair. In the opposite flank, we injected cells from each of Activation of Apoptosis by 4E-BP1 in Fibroblasts Expressing the four independently derived CREF/Ras/BP1-wt clones or as a nega- tive control, untransformed CREF. Tumor formation was documented Oncogenic Ras—FRAP/mTOR has a dual function in the regu- photographically and quantified after 15 days by weight. Tumors were lation of translation. It stimulates protein synthesis by regu- fixed in 10% buffered formalin overnight, processed for routine histol- lating ribosomal biogenesis through p70s6k (22) and specifi- ogy, and examined by a pathologist (JCM) in a blinded fashion. cally activates cap-dependent translation by phosphorylating Statistics—Results of flow cytometry were tabulated as the mean 6 the 4E-BPs (8 –10). Our previous work linking suppression of S.D. of two to five separate experiments. In each experiment, all con- ditions were examined in duplicate or triplicate. For analysis of tumor- apoptosis to the cap-dependent translation initiation appara- igenicity, mitotic and apoptotic indices represent the average number of tus (15) led us to explore whether the 4E-BPs modulate Ras- events/3600 microscopic field (quantified in 10 fields); and tumor dependent viability and chemoresistance. CREF/RasV12 and weights (CREF/RasV12 versus CREF/Ras/BP1 clones) were compared CREF cells were transfected with BP1-wt linked to a puromy- using a paired t test on a log scale. cin selectable marker or with vector carrying only the select- RESULTS able marker, and puromycin-resistant clones were isolated. Activation of Apoptosis in Ras-transformed Fibroblasts by Four CREF/Ras/BP1-wt and four CREF/BP1-wt clonal lines Rapamycin—We used a cell system (21) in which constitutively were developed and assayed for steady state levels of 4E-BP1. 24778 Translational Repressor 4E-BP1 Inhibits Ras Survival Signaling of 4E-BP1 in CREF/RasV12 was directly related to its ability to sequester eIF4E from the translationally active eIF4E-eIF4GI complex, cellular extracts from each clonal line of CREF/Ras/ BP1-wt were incubated with the cap analogue m GTP-agarose to capture eIF4E and its cellular binding partners. The levels of cap-bound eIF4E, 4E-BP1, and eIF4GI were quantified by im- munoblotting and densitometry. Each CREF/RasV12/BP1-wt clone displayed eIF4E associated with significantly increased amounts of fast migrating, hypophosphorylated 4E-BP1 (Fig. 3a). Consistent with this, clones ectopically expressing 4E- BP1-wt generally manifested decreased amounts of eIF4GI bound to eIF4E. Although clone 2 revealed relatively high levels of eIF4GI, there was also an increased amount of eIF4E in the m GTP-captured material. Thus, the ratio of eIF4GI to cap analogue-bound eIF4E was significantly decreased in all 4E-BP1 clones tested, confirming the ability of overexpressed 4E-BP1 to inhibit assembly of the eIF4F translation pre-initi- ation complex. The apoptotic frequency in clones co-expressing activated Ras and 4E-BP1 was proportional to the amount of 4E-BP1 complexed with eIF4E (Fig. 3b) and was inversely related to the eIF4GI/eIF4E ratio (Fig. 3c), a relationship ob- served in the presence and absence of lovastatin. Thus, stimu- lation of apoptotic death by 4E-BP1 was a function of its activ- ity in competitively displacing eIF4GI from eIF4E. To determine whether the interaction of 4E-BP1 with elF4E was a strict requirement for the proapoptotic function of 4E- BP1 in Ras-transformed cells, we utilized a 4E-BP1 deletion mutant (4E-BP1-D), which lacks the eIF4E binding site (18). Transient transfection of CREF/RasV12 with 4E-BP1-wt en- hanced spontaneous apoptosis and sensitized cells to lovastatin in a manner similar to that observed in the stable CREF/ RasV12/BP1-wt clones, suggesting that activation of apoptosis in 4E-BP1-transfected clones was not due to secondary genetic changes during clonal selection (Fig. 3d). In marked contrast, transient transfection with 4E-BP1-D had minimal effects on viability, despite similar levels of 4E-BP1 expression. Thus, the FIG.2. 4E-BP1 sensitizes Ras-transformed cells to apoptosis. ability of 4E-BP1 to bind eIF4E was essential for its blockade of Apoptosis was quantified by flow cytometry (a and b) and visualized by Ras-induced survival signaling. acridine orange staining (c and d). Apoptosis and immunoblot analysis Effect of 4E-BP1 on Apoptosis of Ras-transformed Fibroblasts of 4E-BP1 expression in clonal cell lines of CREF/Ras/V12 (a) and CREF (b) transfected with a construct encoding wild type 4E-BP1 are shown. in Vivo—Prior studies have shown that ectopic 4E-BP1 decreases Cells were cultured for 24 h in the presence (closed circles) or absence the mitotic index and tumorigenicity of NIH 3T3 cells trans- (open circles)of5 mM lovastatin. Each point represents the mean 6 S.D. formed with either eIF4E or src (23); apoptosis was not evalu- (n 5 3) (c and d). Micrographs of Ras-transformed (c) and nontrans- ated. To study all three parameters in Ras-transformed fibro- formed CREF (d) expressing wild type 4E-BP1 (3300) or puromycin vector (shown in the inset, 375). blasts, we injected cells from the CREF/Ras/BP1 clonal lines into immunodeficient mice. Tumors formed by each CREF/Ras/BP1 Under conditions in which expression of endogenous 4E-BP1 line tested were less than one-third the size of those formed by in all mock-transfected cells was undetectable, BP1-wt-trans- mock-transfected CREF/Ras V12, with less visible vascularity fected clones displayed a range of ectopic 4E-BP1 expression. (Fig. 4, a and b). All CREF/RasV12 tumors contained cells form- Western blot analysis performed on total cellular extracts re- ing ill-defined fascicles with ovoid nuclei and an elongated cyto- vealed human 4E-BP1 represented by hypo-(a), intermediate plasm; apoptotic cells were rarely observed (Fig. 4c). In contrast, (b), and hyperphosphorylated (g) forms (9, 16) (Fig. 2a), with tumors formed by cells ectopically expressing 4E-BP1 displayed the a form appearing as a doublet in some of the clones. Quan- more nuclear pleomorphism and most microscopic fields con- tification of apoptosis by flow cytometry revealed that ectopic tained scattered apoptotic cells (Fig. 4, d and e). Ectopic 4E-BP1 4E-BP1 significantly increased the rate of spontaneous apo- decreased the mitotic index of the tumor cells by approximately ptosis in Ras-transformed cells in a dose-dependent manner. one-third and dramatically increased their apoptotic frequency This 2– 8-fold augmentation in basal apoptotic frequency was by nearly 5-fold (Fig. 4f). Untransformed CREF did not form approximately doubled in the presence of lovastatin (Fig. 2a). tumors. These findings establish that suppression of apoptosis in In sharp contrast to the results with transformed CREF/ Ras-transformed cells in vivo depends in part on cap-dependent RasV12, 4E-BP1 did not activate apoptosis in nontransformed translation, a function that was not rescued by transcriptional or parental CREF lacking activated Ras (Fig. 2b). Whereas many post-translational processes. cells comprising the CREF/Ras/BP1-wt clonal lines displayed DISCUSSION morphological hallmarks of apoptosis (Fig. 2c), ectopic expres- sion of 4E-BP1 did not alter the morphology of CREF (Fig. 2d). For nearly four decades, global translational control has been Thus, ectopic 4E-BP1 shifted Ras signaling from suppression to recognized as a fundamental regulatory process in biology (24). induction of apoptosis. More recently, examples of selective control have emerged involv- Relationship between Apoptosis and Sequestration of eIF4E ing regulation at the translation initiation step, particularly in by 4E-BP1—To investigate whether the proapoptotic function the integration of pleiotropic responses leading to differentiation, Translational Repressor 4E-BP1 Inhibits Ras Survival Signaling 24779 FIG.3. 4E-BP1-promoted apoptosis in Ras-transformed cells is associ- ated with displacement of transla- tion factor eIF4GI from eIF4E. a, im- munoblot analysis of 4E-BP1 and eIF4GI associated with cap-bound eIF4E in clones of CREF/RasV12 ectopically ex- pressing wild type 4E-BP1. b and c, apo- ptosis is shown as a function of the 4E- BP1/eIF4E (b) or eIF4G/eIF4E (c) ratio in the indicated CREF/Ras/BP1-wt clones incubated in growth medium for 24 h in the presence (closed circles) or absence (open circles)of5 mM lovastatin. d, 4E- BP1 lacking an eIF4E binding domain does not promote apoptosis. Shown are nonspecific green fluorescence (open his- tograms), expression of HA (closed histo- grams), and DNA content (shaded histo- grams) in CREF/RasV12 transfected with an empty HA vector, an HA-tagged wild type 4E-BP1, or an HA-tagged eIF4E binding site deletion mutant, 4E-BP1-D. The results of a representative experi- ment are shown (three independent transfection experiments yielded similar results). proliferation, and survival (25, 26). Here we focus on the trans- chinery subserves an important role in the regulation of apo- lational apparatus itself, examining initiation events involving ptosis (15, 27, 28). In our study design, we experimentally the mRNA cap-binding protein eIF4E and its most abundant separate the viability effects of global versus cap-dependent repressor, 4E-BP1. We find that concentrations of rapamycin translation. We find here that up to an 80% reduction of global that are known to inhibit 4E-BP1 phosphorylation and cap-de- protein synthesis with cycloheximide actually blocks apoptosis, pendent protein synthesis (10) sensitize fibroblasts carrying ac- whereas similar levels of translational repression with the tivated Ras to apoptosis, whereas nonselective inhibition of glo- FRAP/mTOR inhibitor rapamycin or ectopic expression of the bal protein synthesis by the peptide elongation inhibitor cap-specific repressor 4E-BP1 have a profound proapoptotic cycloheximide actually blocks apoptosis. We further show that effect. The importance of cap-dependent protein synthesis in enforced expression of 4E-BP1 in Ras-transformed fibroblasts viability regulation is further supported by work demonstrat- activates apoptosis, eliminates resistance to cytostatic drugs, and ing that the translation initiation factor eIF4G is cleaved early inhibits tumorigenicity. In contrast, cell viability is unaltered in the process of apoptosis (29 –31) leading to a shut off of when 4E-BP1 is ectopically expressed in nontransformed cells. cap-dependent protein synthesis. In addition, recent reports The proapoptotic activity of 4E-BP1 is strictly dependent on its implicate cap-independent translation through internal riboso- ability to sequester the mRNA cap-binding protein, eIF4E, thus mal entry sites in the synthesis of some proapoptotic proteins preventing assembly of an active pre-initiation translation com- (27, 32), and for Myc where detailed studies have been carried plex. These results add cap-dependent translation to the estab- out, translation is sustained even during apoptosis by initiation lished transcriptional and post-translational mechanisms in- utilizing an internal ribosomal entry site (32). volved in the regulation of apoptosis by oncogenic Ras and The downstream effector proteins linking the cap-dependent identify a translationally regulated step as essential for Ras-de- translation initiation apparatus to the apoptotic machinery pendent drug resistance. and the precise mechanisms regulating the translation of their Mounting evidence now suggests that the translational ma- cognate mRNAs are unknown. Prior studies have identified 24780 Translational Repressor 4E-BP1 Inhibits Ras Survival Signaling notypic changes (38), and here we find that even dramatic overexpression in nontransformed fibroblasts is compatible with physiological function. Against the background of onco- genic Ras, however, 4E-BP1 exerts powerful control over cell growth, viability, and susceptibility to cytostatic drugs. These findings suggest that translational repressors may constitute a significant component of the mammalian tumor surveillance system. In addition, our work identifies a novel mechanism whereby tumor cells bearing oncogenic Ras can acquire resist- ance to genotoxic and nongenotoxic therapeutic agents. Our data thus provide direct evidence linking the fundamental bi- ological process of cap-dependent translation initiation with suppression of apoptosis by activated Ras. Acknowledgments—We thank A. De Benedetti for cell lines and dis- cussion, P. Jolicoeur for the pSRa vector, J. Geagea for help cloning pSRa-BPI, J. Murray and Darlene Charboneau for technical assistance, the University of Minnesota Cancer Center Biostatistical Core for as- sistance in study design and data analysis, and B. Raught for critical review of the manuscript. 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Translational Control of the Antiapoptotic Function of Ras

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 32, Issue of August 11, pp. 24776 –24780, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Received for publication, March 7, 2000, and in revised form, April 28, 2000 Published, JBC Papers in Press, May 12, 2000, DOI 10.1074/jbc.M001938200 Vitaly A. Polunovsky‡, Anne-Claude Gingras§, Nahum Sonenberg§, Mark Peterson‡, Annie Tan‡, Jeffrey B. Rubins‡, J. Carlos Manivel¶, and Peter B. Bitterman‡i From the ‡Department of Medicine and the ¶Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota 55455 and the §Department of Biochemistry, McGill University, Montreal, Quebec H3G IY6, Canada Activated Ras has been shown to provide powerful the intrinsic apoptotic machinery. Akt also regulates the FK506 binding-protein 12 (FKBP12)-rapamycin-associated antiapoptotic signals to cells through well defined tran- scriptional and post- translational pathways, whereas protein/mammalian target of rapamycin (FRAP/mTOR), a ki- translational control as a mechanism of Ras survival nase which functions in the control of translation by activating signaling remains unexplored. Here we show a direct two components of the protein synthesis apparatus: (i) the relationship between assembly of the cap-dependent initiation complex binding the 59-mRNA cap and (ii) the 40 S s6k translation initiation apparatus and suppression of apo- ribosomal protein S6 kinase, p70 (8 –11). However, data ptosis by oncogenic Ras in vitro and in vivo. Decreasing examining the importance of translational control in the mech- protein synthesis with rapamycin, which is known to anism of Ras survival signaling are lacking. inhibit cap-dependent translation, increases the suscep- Translational control is usually exerted at the initiation step. tibility of Ras-transformed fibroblasts to cytostatic In eukaryotes, the 59-mRNA cap is bound by the eukaryotic drug-induced apoptosis. In contrast, suppressing global translation initiation complex eIF4F, which consists of a bi- protein synthesis with equipotent concentrations of cy- directional RNA helicase eIF4A, the docking protein eIF4G and cloheximide actually prevents apoptosis. Enforced ex- the cap binding subunit eIF4E (12). A major target for regula- pression of the cap-dependent translational repressor, tion, eIF4E is considered to be rate limiting for translation the eukaryotic translation initiation factor (eIF) 4E- initiation under most circumstances (13), and its up-regulation binding protein (4E-BPI), sensitizes fibroblasts to apo- is associated with cell proliferation, suppression of apoptosis, ptosis in a manner strictly dependent on its ability to and tumorigenicity (14, 15). The function of eIF4E is inhibited sequester eIF4E from a translationally active complex by members of the family of translational repressors, the with eIF4GI and the co-expression of oncogenic Ras. eIF4E-binding proteins (4E-BPs, also known as PHAS) (13, 16). Ectopic expression of 4E-BP1 also promotes apoptosis of When hypophosphorylated, 4E-BPs compete with eIF4G for Ras-transformed cells injected into immunodeficient binding to eIF4E and sequester eIF4E in a nonfunctional com- mice and markedly diminishes their tumorigenicity. These results establish that eIF4E-dependent protein plex. Upon hyperphosphorylation, 4E-BPs dissociate from the synthesis is essential for survival of fibroblasts bearing complex with eIF4E allowing it to form an active translation oncogenic Ras and support the concept that activation initiation complex (16, 17). To elucidate the role of transla- of cap-dependent translation by extracellular ligands or tional control in Ras survival signaling, here we focus directly intrinsic survival signaling molecules suppresses apo- on the 59-mRNA cap binding complex and examine the induc- ptosis, whereas synthesis of proteins mediating apo- tion of apoptosis in cells transformed with oncogenic Ras after ptosis can occur independently of the cap. a generalized reduction in protein synthesis or after specific repression of cap-dependent translation initiation. EXPERIMENTAL PROCEDURES Extracellular survival factors suppress the intrinsic apo- Generation of Clones and Transient Transfection—Cloned rat em- ptotic apparatus through cognate receptor kinases at the cell bryo fibroblasts (CREF) and CREF/RasV12 (a gift from A. De Benedetti) surface, which activate the proto-oncogene ras and a number of were subcloned and maintained in Dulbecco’s modified Eagle’s medium pleiotropic transcriptional and post-translational effector path- supplemented with 10% fetal calf serum. The coding sequence of human ways (1). A major effector of Ras survival signaling is the 4E-BP1 was amplified by polymerase chain reaction and directionally serine/threonine protein kinase, Akt (2, 3). Transcriptional con- cloned into the EcoRI and BamHI sites of the mammalian expression trol is exerted by Akt-mediated phosphorylation of the Fork- vector pSRapuro, (a kind gift from Dr. P. Jolicoeur, Institut de Recher- ches Cliniques, Montreal). To generate the eIF4E binding site deletion head1 family transcription factor FKHRL1(4) and the tran- mutant (amino acids 54 – 63; 4E-BP1-D), the 4E-BP1 coding sequence scription factor nuclear factor-kB (5), which alter expression of was cloned into the cytomegalovirus-based vector pACTAG-2, which apoptosis-related genes. Akt-mediated phosphorylation of the was used as a template for polymerase chain reaction site-directed Bcl-2 family member Bad (6) and the cell death protease mutagenesis (18). Clones of CREF and CREF/RasV12 expressing wild caspase-9 (7) is implicated in post-translational suppression of type or mutant 4E-BP1 were generated using the FuGENE 6 (Roche Diagnostics) transfection technique. Selection of transfected cells was begun after 24 h with medium containing 1 mg/ml puromycin, and * This work was supported by NHLBI, National Institutes of Health- resistant clones were isolated after 12–16 days. CREF/RasV12s were funded SCOR Grant 2P50-HL50152, a grant from the National Cancer also transiently transfected with a pACTAG-2 construct encoding he- Institute of Canada, a doctoral award from the Medical Research Coun- magglutinin (HA)-tagged human 4E-BP1-wt, 4E-BP1-D, or a vector cil of Canada, and the M.D. Ph.D. program of the University of Minne- carrying only the HA tag. To detect the level of HA expression by flow sota Medical School. The costs of publication of this article were de- frayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 The abbreviations used are: FRAP/mTOR, FKBP12-rapamycin-as- U.S.C. Section 1734 solely to indicate this fact. sociated protein/mammalian target of rapamycin; eIF, lukaryotic trans- To whom correspondence should be addressed. Tel.: 612-624-0999; lation initiation factor; 4E-BP, eIF4E-binding protein; CREF, cloned rat Fax: 612-625-2174; E-mail: [email protected].. embryo fibroblasts; HA, hemagglutinin; BP1-wt, wild type 4E-BP1. 24776 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Translational Repressor 4E-BP1 Inhibits Ras Survival Signaling 24777 FIG.1. Rapamycin suppresses Ras- induced chemoresistance. a, expres- sion of Ras protein in CREF/Neo and CREF/RasV12. b, apoptosis was assessed after incubation (24 h, 37 °C) with or without 75 nM rapamycin in medium alone (solid bar) or medium supple- mented either with 5 mM lovastatin (striped bar)or75nM camptothecin (open bar). Values shown represent the mean 6 S.D. (n 5 3) of the percentage of cells with subdiploid DNA content. c and d, lovasta- tin-induced apoptosis in association with suppression of protein synthesis by ra- pamycin or cycloheximide. The rates of protein synthesis and lovastatin-induced apoptosis were quantified in parallel cul- tures exposed for 24 h to different con- centrations of either rapamycin (c)or cycloheximide (d). Protein synthesis is displayed as a percent of that observed without inhibitors. cytometry, cells were fixed with absolute ethanol and incubated for 16 h expressed oncogenic RasV12 enables CREF to survive in oth- at 4 °C with mouse anti-HA IgG2 antibody (4 mg/ml, Roche Molecular bk erwise lethal concentrations of cytostatic drugs (nongenotoxic, Biochemicals) or with mouse isotype-specific IgG2 antibody (4 mg/ml, bk lovastatin; genotoxic, camptothecin, Fig. 1). The FRAP/mTOR PharMingen) followed by incubation with fluorescein-conjugated anti- inhibitor rapamycin completely abrogated Ras-dependent re- mouse IgG antibody (1:40, Sigma) for 30 min. Immunoblot Analysis of Cap-bound Proteins—Cell lysates (250 ml sistance to drug-induced cell death (Fig. 1b) and even when containing 250 mg of protein) were incubated with m GTP-Sepharose applied as a single agent, stimulated apoptosis in cells express- resin (Amersham Pharmacia Biotech) to capture eIF4E and its binding ing activated Ras. This proapoptotic effect of rapamycin was partners (9). Samples were eluted with buffer containing 70 mM m GTP. not observed in nontransformed fibroblasts. These observa- Cap-bound material was subjected to SDS-polyacrylamide gel electro- tions confirm a dual proapoptotic and antiapoptotic function for phoresis and transferred to nitrocellulose. Blots were probed first for RasV12 (2) and implicate FRAP/mTOR in Ras-dependent res- eIF4E (mouse monoclonal antibody, 1:500, Transduction Laboratories), then stripped and probed for 4E-BP1 (rabbit polyclonal antiserum cue from both Ras-activated and drug-triggered apoptotic 1:2500) (19), and stripped and probed a third time for eIF4GI (rabbit pathways. polyclonal antibody 1:4000) (20) When rapamycin was added to Ras-transformed cells, it Apoptosis Assays—Frequency of apoptosis was quantified by flow caused a dose-dependent decline in protein synthesis, which cytometric analysis of the percentage of cells with hypodiploid DNA paralleled its ability to sensitize cells to lovastatin-induced content. Adherent and nonadherent cells were pooled, washed in phos- phate-buffered saline, and fixed with ice-cold 70% ethanol for at least apoptosis (Fig. 1c). Of note, equipotent doses of the peptide 1 h. Fixed cells were washed and incubated in propidium iodide stain elongation inhibitor cycloheximide actually blocked apoptosis mixture 50 mg/ml propidium iodide, 0.05% Triton X-100, 37 mg/ml (Fig. 1d), demonstrating that the execution of lovastatin-in- EDTA, 100 units/ml ribonuclease in phosphate-buffered saline). After duced cell death requires global protein synthesis. These re- incubation for 45 min at 37 °C, DNA content was determined by quan- sults suggest that a generalized inhibition of mRNA transla- titative flow cytometry using standard optics of the FACScan flow cytometer (Becton Dickinson) and the CellQuest program. tion is not the means by which rapamycin exerts its Tumorigenicity Assay—Under sterile conditions, 3 3 10 cells in proapoptotic effect, rather they point toward a selective inhi- phosphate-buffered saline were injected into each flank of immunodefi- bition of antiapoptotic mRNA translation or a mechanism in- cient mice (Nu Nu, Harlan). CREF/RasV12 cells were injected into one dependent of its ability to repress translation. flank of each pair. In the opposite flank, we injected cells from each of Activation of Apoptosis by 4E-BP1 in Fibroblasts Expressing the four independently derived CREF/Ras/BP1-wt clones or as a nega- tive control, untransformed CREF. Tumor formation was documented Oncogenic Ras—FRAP/mTOR has a dual function in the regu- photographically and quantified after 15 days by weight. Tumors were lation of translation. It stimulates protein synthesis by regu- fixed in 10% buffered formalin overnight, processed for routine histol- lating ribosomal biogenesis through p70s6k (22) and specifi- ogy, and examined by a pathologist (JCM) in a blinded fashion. cally activates cap-dependent translation by phosphorylating Statistics—Results of flow cytometry were tabulated as the mean 6 the 4E-BPs (8 –10). Our previous work linking suppression of S.D. of two to five separate experiments. In each experiment, all con- ditions were examined in duplicate or triplicate. For analysis of tumor- apoptosis to the cap-dependent translation initiation appara- igenicity, mitotic and apoptotic indices represent the average number of tus (15) led us to explore whether the 4E-BPs modulate Ras- events/3600 microscopic field (quantified in 10 fields); and tumor dependent viability and chemoresistance. CREF/RasV12 and weights (CREF/RasV12 versus CREF/Ras/BP1 clones) were compared CREF cells were transfected with BP1-wt linked to a puromy- using a paired t test on a log scale. cin selectable marker or with vector carrying only the select- RESULTS able marker, and puromycin-resistant clones were isolated. Activation of Apoptosis in Ras-transformed Fibroblasts by Four CREF/Ras/BP1-wt and four CREF/BP1-wt clonal lines Rapamycin—We used a cell system (21) in which constitutively were developed and assayed for steady state levels of 4E-BP1. 24778 Translational Repressor 4E-BP1 Inhibits Ras Survival Signaling of 4E-BP1 in CREF/RasV12 was directly related to its ability to sequester eIF4E from the translationally active eIF4E-eIF4GI complex, cellular extracts from each clonal line of CREF/Ras/ BP1-wt were incubated with the cap analogue m GTP-agarose to capture eIF4E and its cellular binding partners. The levels of cap-bound eIF4E, 4E-BP1, and eIF4GI were quantified by im- munoblotting and densitometry. Each CREF/RasV12/BP1-wt clone displayed eIF4E associated with significantly increased amounts of fast migrating, hypophosphorylated 4E-BP1 (Fig. 3a). Consistent with this, clones ectopically expressing 4E- BP1-wt generally manifested decreased amounts of eIF4GI bound to eIF4E. Although clone 2 revealed relatively high levels of eIF4GI, there was also an increased amount of eIF4E in the m GTP-captured material. Thus, the ratio of eIF4GI to cap analogue-bound eIF4E was significantly decreased in all 4E-BP1 clones tested, confirming the ability of overexpressed 4E-BP1 to inhibit assembly of the eIF4F translation pre-initi- ation complex. The apoptotic frequency in clones co-expressing activated Ras and 4E-BP1 was proportional to the amount of 4E-BP1 complexed with eIF4E (Fig. 3b) and was inversely related to the eIF4GI/eIF4E ratio (Fig. 3c), a relationship ob- served in the presence and absence of lovastatin. Thus, stimu- lation of apoptotic death by 4E-BP1 was a function of its activ- ity in competitively displacing eIF4GI from eIF4E. To determine whether the interaction of 4E-BP1 with elF4E was a strict requirement for the proapoptotic function of 4E- BP1 in Ras-transformed cells, we utilized a 4E-BP1 deletion mutant (4E-BP1-D), which lacks the eIF4E binding site (18). Transient transfection of CREF/RasV12 with 4E-BP1-wt en- hanced spontaneous apoptosis and sensitized cells to lovastatin in a manner similar to that observed in the stable CREF/ RasV12/BP1-wt clones, suggesting that activation of apoptosis in 4E-BP1-transfected clones was not due to secondary genetic changes during clonal selection (Fig. 3d). In marked contrast, transient transfection with 4E-BP1-D had minimal effects on viability, despite similar levels of 4E-BP1 expression. Thus, the FIG.2. 4E-BP1 sensitizes Ras-transformed cells to apoptosis. ability of 4E-BP1 to bind eIF4E was essential for its blockade of Apoptosis was quantified by flow cytometry (a and b) and visualized by Ras-induced survival signaling. acridine orange staining (c and d). Apoptosis and immunoblot analysis Effect of 4E-BP1 on Apoptosis of Ras-transformed Fibroblasts of 4E-BP1 expression in clonal cell lines of CREF/Ras/V12 (a) and CREF (b) transfected with a construct encoding wild type 4E-BP1 are shown. in Vivo—Prior studies have shown that ectopic 4E-BP1 decreases Cells were cultured for 24 h in the presence (closed circles) or absence the mitotic index and tumorigenicity of NIH 3T3 cells trans- (open circles)of5 mM lovastatin. Each point represents the mean 6 S.D. formed with either eIF4E or src (23); apoptosis was not evalu- (n 5 3) (c and d). Micrographs of Ras-transformed (c) and nontrans- ated. To study all three parameters in Ras-transformed fibro- formed CREF (d) expressing wild type 4E-BP1 (3300) or puromycin vector (shown in the inset, 375). blasts, we injected cells from the CREF/Ras/BP1 clonal lines into immunodeficient mice. Tumors formed by each CREF/Ras/BP1 Under conditions in which expression of endogenous 4E-BP1 line tested were less than one-third the size of those formed by in all mock-transfected cells was undetectable, BP1-wt-trans- mock-transfected CREF/Ras V12, with less visible vascularity fected clones displayed a range of ectopic 4E-BP1 expression. (Fig. 4, a and b). All CREF/RasV12 tumors contained cells form- Western blot analysis performed on total cellular extracts re- ing ill-defined fascicles with ovoid nuclei and an elongated cyto- vealed human 4E-BP1 represented by hypo-(a), intermediate plasm; apoptotic cells were rarely observed (Fig. 4c). In contrast, (b), and hyperphosphorylated (g) forms (9, 16) (Fig. 2a), with tumors formed by cells ectopically expressing 4E-BP1 displayed the a form appearing as a doublet in some of the clones. Quan- more nuclear pleomorphism and most microscopic fields con- tification of apoptosis by flow cytometry revealed that ectopic tained scattered apoptotic cells (Fig. 4, d and e). Ectopic 4E-BP1 4E-BP1 significantly increased the rate of spontaneous apo- decreased the mitotic index of the tumor cells by approximately ptosis in Ras-transformed cells in a dose-dependent manner. one-third and dramatically increased their apoptotic frequency This 2– 8-fold augmentation in basal apoptotic frequency was by nearly 5-fold (Fig. 4f). Untransformed CREF did not form approximately doubled in the presence of lovastatin (Fig. 2a). tumors. These findings establish that suppression of apoptosis in In sharp contrast to the results with transformed CREF/ Ras-transformed cells in vivo depends in part on cap-dependent RasV12, 4E-BP1 did not activate apoptosis in nontransformed translation, a function that was not rescued by transcriptional or parental CREF lacking activated Ras (Fig. 2b). Whereas many post-translational processes. cells comprising the CREF/Ras/BP1-wt clonal lines displayed DISCUSSION morphological hallmarks of apoptosis (Fig. 2c), ectopic expres- sion of 4E-BP1 did not alter the morphology of CREF (Fig. 2d). For nearly four decades, global translational control has been Thus, ectopic 4E-BP1 shifted Ras signaling from suppression to recognized as a fundamental regulatory process in biology (24). induction of apoptosis. More recently, examples of selective control have emerged involv- Relationship between Apoptosis and Sequestration of eIF4E ing regulation at the translation initiation step, particularly in by 4E-BP1—To investigate whether the proapoptotic function the integration of pleiotropic responses leading to differentiation, Translational Repressor 4E-BP1 Inhibits Ras Survival Signaling 24779 FIG.3. 4E-BP1-promoted apoptosis in Ras-transformed cells is associ- ated with displacement of transla- tion factor eIF4GI from eIF4E. a, im- munoblot analysis of 4E-BP1 and eIF4GI associated with cap-bound eIF4E in clones of CREF/RasV12 ectopically ex- pressing wild type 4E-BP1. b and c, apo- ptosis is shown as a function of the 4E- BP1/eIF4E (b) or eIF4G/eIF4E (c) ratio in the indicated CREF/Ras/BP1-wt clones incubated in growth medium for 24 h in the presence (closed circles) or absence (open circles)of5 mM lovastatin. d, 4E- BP1 lacking an eIF4E binding domain does not promote apoptosis. Shown are nonspecific green fluorescence (open his- tograms), expression of HA (closed histo- grams), and DNA content (shaded histo- grams) in CREF/RasV12 transfected with an empty HA vector, an HA-tagged wild type 4E-BP1, or an HA-tagged eIF4E binding site deletion mutant, 4E-BP1-D. The results of a representative experi- ment are shown (three independent transfection experiments yielded similar results). proliferation, and survival (25, 26). Here we focus on the trans- chinery subserves an important role in the regulation of apo- lational apparatus itself, examining initiation events involving ptosis (15, 27, 28). In our study design, we experimentally the mRNA cap-binding protein eIF4E and its most abundant separate the viability effects of global versus cap-dependent repressor, 4E-BP1. We find that concentrations of rapamycin translation. We find here that up to an 80% reduction of global that are known to inhibit 4E-BP1 phosphorylation and cap-de- protein synthesis with cycloheximide actually blocks apoptosis, pendent protein synthesis (10) sensitize fibroblasts carrying ac- whereas similar levels of translational repression with the tivated Ras to apoptosis, whereas nonselective inhibition of glo- FRAP/mTOR inhibitor rapamycin or ectopic expression of the bal protein synthesis by the peptide elongation inhibitor cap-specific repressor 4E-BP1 have a profound proapoptotic cycloheximide actually blocks apoptosis. We further show that effect. The importance of cap-dependent protein synthesis in enforced expression of 4E-BP1 in Ras-transformed fibroblasts viability regulation is further supported by work demonstrat- activates apoptosis, eliminates resistance to cytostatic drugs, and ing that the translation initiation factor eIF4G is cleaved early inhibits tumorigenicity. In contrast, cell viability is unaltered in the process of apoptosis (29 –31) leading to a shut off of when 4E-BP1 is ectopically expressed in nontransformed cells. cap-dependent protein synthesis. In addition, recent reports The proapoptotic activity of 4E-BP1 is strictly dependent on its implicate cap-independent translation through internal riboso- ability to sequester the mRNA cap-binding protein, eIF4E, thus mal entry sites in the synthesis of some proapoptotic proteins preventing assembly of an active pre-initiation translation com- (27, 32), and for Myc where detailed studies have been carried plex. These results add cap-dependent translation to the estab- out, translation is sustained even during apoptosis by initiation lished transcriptional and post-translational mechanisms in- utilizing an internal ribosomal entry site (32). volved in the regulation of apoptosis by oncogenic Ras and The downstream effector proteins linking the cap-dependent identify a translationally regulated step as essential for Ras-de- translation initiation apparatus to the apoptotic machinery pendent drug resistance. and the precise mechanisms regulating the translation of their Mounting evidence now suggests that the translational ma- cognate mRNAs are unknown. Prior studies have identified 24780 Translational Repressor 4E-BP1 Inhibits Ras Survival Signaling notypic changes (38), and here we find that even dramatic overexpression in nontransformed fibroblasts is compatible with physiological function. Against the background of onco- genic Ras, however, 4E-BP1 exerts powerful control over cell growth, viability, and susceptibility to cytostatic drugs. These findings suggest that translational repressors may constitute a significant component of the mammalian tumor surveillance system. In addition, our work identifies a novel mechanism whereby tumor cells bearing oncogenic Ras can acquire resist- ance to genotoxic and nongenotoxic therapeutic agents. Our data thus provide direct evidence linking the fundamental bi- ological process of cap-dependent translation initiation with suppression of apoptosis by activated Ras. Acknowledgments—We thank A. De Benedetti for cell lines and dis- cussion, P. Jolicoeur for the pSRa vector, J. Geagea for help cloning pSRa-BPI, J. Murray and Darlene Charboneau for technical assistance, the University of Minnesota Cancer Center Biostatistical Core for as- sistance in study design and data analysis, and B. Raught for critical review of the manuscript. 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Published: Aug 1, 2000

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