ERK and p38 Inhibit the Expression of 4E-BP1 Repressor of Translation through Induction of Egr-1
2003; Elsevier BV; Volume: 278; Issue: 21 Linguagem: Inglês
10.1074/jbc.m211696200
ISSN1083-351X
AutoresMalvyne Rolli‐Derkinderen, François Machavoine, Jay M. Baraban, Annabelle Grolleau, Laura Beretta, Michel Dy,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
Resumo4E-BP1 plays a major role in translation by inhibiting cap-dependent translation initiation. Several reports have investigated the regulation of 4E-BP1 phosphorylation, which varies along with cell differentiation and upon various stimulations, but very little is known about the regulation of its expression. In a first part, we show that the expression of 4E-BP1 protein and transcript decreases in hematopoietic cell lines cultivated in the presence of phorbol 12-myristate 13-acetate (PMA). This decrease depends on the activation of the ERK/mitogen-activated protein kinases. 4E-BP1 expression also decreases when the p38/mitogen-activated protein kinase pathway is activated by granulocyte/macrophage colony-stimulating factor but to a lesser extent than with PMA. In a second part, we examine how 4e-bp1 promoter activity is regulated. PMA and granulocyte/macrophage colony-stimulating factor induce Egr-1 expression through ERK and p38 activation, respectively. Using a dominant negative mutant of Egr, ZnEgr, we show that this transcription factor is responsible for the inhibition of 4e-bp1 promoter activity. In a third part we show that histidine decarboxylase, whose activity and expression are inversely correlated with 4E-BP1 expression, is a potential target for the translational machinery. These data (i) are the first evidence of a new role of ERK and p38 on the translational machinery and (ii) demonstrate that 4E-BP1 is a new target for Egr-1. 4E-BP1 plays a major role in translation by inhibiting cap-dependent translation initiation. Several reports have investigated the regulation of 4E-BP1 phosphorylation, which varies along with cell differentiation and upon various stimulations, but very little is known about the regulation of its expression. In a first part, we show that the expression of 4E-BP1 protein and transcript decreases in hematopoietic cell lines cultivated in the presence of phorbol 12-myristate 13-acetate (PMA). This decrease depends on the activation of the ERK/mitogen-activated protein kinases. 4E-BP1 expression also decreases when the p38/mitogen-activated protein kinase pathway is activated by granulocyte/macrophage colony-stimulating factor but to a lesser extent than with PMA. In a second part, we examine how 4e-bp1 promoter activity is regulated. PMA and granulocyte/macrophage colony-stimulating factor induce Egr-1 expression through ERK and p38 activation, respectively. Using a dominant negative mutant of Egr, ZnEgr, we show that this transcription factor is responsible for the inhibition of 4e-bp1 promoter activity. In a third part we show that histidine decarboxylase, whose activity and expression are inversely correlated with 4E-BP1 expression, is a potential target for the translational machinery. These data (i) are the first evidence of a new role of ERK and p38 on the translational machinery and (ii) demonstrate that 4E-BP1 is a new target for Egr-1. Control of mRNA translation plays a pivotal role in regulating gene expression under a variety of conditions in mammalian cells (1Schneider R. Agol V.I. Andino R. Bayard F. Cavener D.R. Chappell S.A. Chen J.J. Darlix J.L. Dasgupta A. Donze O. Duncan R. Elroy-Stein O. Farabaugh P.J. Filipowicz W. Gale Jr., M. Gehrke L. Goldman E. Groner Y. Harford J.B. Hatzglou M. He B. Hellen C.U. Hentze M.W. Hershey J. Hershey P. Hohn T. Holcik M. Hunter C.P. Igarashi K. Jackson R. Jagus R. Jefferson L.S. Joshi B. Kaempfer R. Katze M. Kaufman R.J. Kiledjian M. Kimball S.R. Kimchi A. Kirkegaard K. Koromilas A.E. Krug R.M. Kruys V. Lamphear B.J. Lemon S. Lloyd R.E. Maquat L.E. Martinez-Salas E. Mathews M.B. Mauro V.P. Miyamoto S. Mohr I. Morris D.R. Moss E.G. Nakashima N. Palmenberg A. Parkin N.T. Pe'ery T. Pelletier J. Peltz S. Pestova T.V. Pilipenko E.V. Prats A.C. Racaniello V. Read G.S. Rhoads R.E. Richter J.D. Rivera-Pomar R. Rouault T. Sachs A. Sarnow P. Scheper G.C. Schiff L. Schoenberg D.R. 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EMBO J. 1995; 14: 3828-3834Crossref PubMed Scopus (251) Google Scholar). eIF4E activity is modulated by phosphorylation in response to mitogens, polypeptide hormones, tumor promoters, and growth factors in a mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MNK, MAPK signal-integrating kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; IL, interleukin; CSF, colony-stimulating factor; GM, granulocyte/macrophage; PMA, phorbol 12-myristate 13-acetate; HDC, histidine decarboxylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGFP, enhanced green fluorescent protein; MEK, MAPK/ERK kinase; OA, okadaic acid; CHX, cycloheximide.1The abbreviations used are: MAPK, mitogen-activated protein kinase; MNK, MAPK signal-integrating kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; IL, interleukin; CSF, colony-stimulating factor; GM, granulocyte/macrophage; PMA, phorbol 12-myristate 13-acetate; HDC, histidine decarboxylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGFP, enhanced green fluorescent protein; MEK, MAPK/ERK kinase; OA, okadaic acid; CHX, cycloheximide.-MAPK signal-integrating kinase (MNK) pathway-dependent manner (5Raught B. Gingras A.C. Int. J. Biochem. Cell Biol. 1999; 31: 43-57Crossref PubMed Scopus (246) Google Scholar). In addition to the regulation of its phosphorylation, the activity of eIF4E is tightly controlled through reversible interaction with a family of inhibitory proteins termed 4E-BP (eIF4E-binding proteins). Of the three known proteins (4E-BP1, 4E-BP2, and 4E-BP3), 4E-BP1, also named PHAS-1, is the best characterized. 4E-BP1 specifically inhibits cap-dependent translation by competing with eIF4G for binding to the cap-binding factor eIF4E and consequently preventing the formation of the eIF4F complex (6Haghighat A. Mader S. Pause A. Sonenberg N. EMBO J. 1995; 14: 5701-5709Crossref PubMed Scopus (525) Google Scholar). The affinity of the 4E-BPs to eIF4E depends on their phosphorylation state. 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Induction of HL-60 and U-987 cell differentiation by PMA or interferon-γ into monocytic/macrophages results in a dephosphorylation and consequent activation of 4E-BP1. In contrast induction of HL-60 into the granulocytic differentiation by Me2SO decreases 4E-BP1 expression level, whereas it increases 4E-BP2 expression level (44Grolleau A. Sonenberg N. Wietzerbin J. Beretta L. J. Immunol. 1999; 162: 3491-3497PubMed Google Scholar, 45Grolleau A Wietzerbin J. Beretta L. Leukemia. 2000; 14: 1909-1914Crossref PubMed Scopus (13) Google Scholar). Expression of 4E-BP2 is down-regulated during thymocyte maturation (46Beretta L. Singer N.G. Hinderer R. Gingras A.C. Richardson B. Hanash S.M. Sonenberg N. J. Immunol. 1998; 160: 3269-3273PubMed Google Scholar), but nothing is known about the mechanisms by which 4E-BP expression is modified. We derived from the pluripotent UT7 cell line a subpopulation, the UT7D1 cell line, which in the presence of the growth factor GM-CSF spontaneously expresses the mRNA coding for histidine decarboxylase (HDC). Stimulation of these cells during 24 h with PMA induces a basophil differentiation characterized by an induction of IL-4, IL-6, and IL-13 expression, the presentation of the basophil Bsp-1 antigen, and an increase in histamine production (47Dy M. Pacilio M. Arnould A. Machavoine F. Mayeux P. Hermine O. Bodger M. Schneider E. Exp. Hematol. 1999; 27: 1295-1305Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Whereas the induction of IL-4, IL-6, and IL-13 expression is regulated at the transcriptional level, the HDC expression seems to be regulated at a post-transcriptional level (47Dy M. Pacilio M. Arnould A. Machavoine F. Mayeux P. Hermine O. Bodger M. Schneider E. Exp. Hematol. 1999; 27: 1295-1305Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 48Maeda K. Taniguchi H. Ohno I. Ohtsu H. Yamauchi K. Sakurai E. Tanno Y. Butterfield J.H. Watanabe T. Shirato K. Exp. Hematol. 1998; 26: 325-331PubMed Google Scholar). Thus we examined the activity of the translational initiation machinery in PMA-treated UT7D1 cells. Such a treatment induced a decrease in 4E-BP1 expression, but 4E-BP2 or eIF4E expressions did not change. We also have shown that 4E-BP1 expression is negatively regulated at the transcriptional level by the ERK and p38 via the induction of Egr-1 expression. Ultimately we have provided new insights into the regulation of HDC expression at the translational level.EXPERIMENTAL PROCEDURESCell Culture and Treatments—Human UT7D1, 11OC1, HEL, K562, and HMC1 cells were maintained in RPMI 1640 (Invitrogen) supplemented with 1% 100× l-glutamine, 10% fetal calf serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. 2 ng/ml of recombinant GM-CSF (R&D) was added to the UT7D1 cell line. Passages were performed every 3 or 4 days at a concentration of 2 × 105 cells/ml. 10 μg/ml cycloheximide (Sigma), 10 ng/ml rapamycin, 25 μm LY294002, 3 μm bisindolylmaleimide, 10 μm U0126, 20 μm PD98059, or 10 μm SB203580 (Calbiochem) were added to the medium 30 min before PMA (Sigma) treatment (20 ng/ml) for 30 min to 48 h as indicated.Northern Blot Analysis—Total RNA were extracted by a modified method of Chomczynski and Sacchi (49Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62986) Google Scholar), using TRIzol Reagent (Invitrogen) according to the manufacturer's instruction. 20 μg of total RNA were separated on agarose formaldehyde gels and blotted onto nylon membranes (HybondTM-N+; Amersham Biosciences). The membrane filters were hybridized with [32P]dATP random primed cDNA probes prepared from a partial 4e-bp1 cDNA fragment amplified per PCR (sense primer, 5′-GGACTACAGCACGACCCCCG-3′, and antisense primer, 5′-TGACTCTTCACCGCCCGCCC-3′), a partial 4e-bp2 cDNA fragment amplified per PCR (sense, primer 5′-GGGGGACGGTCTTCTCCACC-3′, and antisense primer, 5′-GCATGTTTCCTGTCGTGATTGTTC-3′), a partial hdc cDNA fragment amplified per PCR (sense primer, 5′-GCCCGATGCTGATGAGTCCT-3′, and antisense primer, 5′-CACCGTCTTCTTCTTAGTCT-3′) or a partial cDNA fragment of egr-1 (GenBankTM accession number AA507023). Hybridized filters were washed under high stringency conditions (0.1× SSC, 70 °C), analyzed by autoradiography, and quantified using QuantityOne. Equal loading of RNA was confirmed by stripping and reprobing the blots with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe or staining of the ribosomal RNA with ethidium bromide.Protein Kinase Assays—1 × 106 UT7D1 cells were washed three times with ice-cold phosphate-buffered saline and harvested in lysis buffer (20 mm Tris acetate, pH 7.0, 0.1 mm EDTA, 1 mm EGTA, 1 mm Na3VO4, 10 mm β-glycerophosphate, 50 mm NaF, 5 mm pyrophosphate, 1% Triton X-100, 1 mm benzamidine, 2 μg/ml leupeptin, 0.1% (v/v) β-mercaptoethanol, 0.27 m sucrose, 0.2 mm phenylmethylsulfonyl fluoride). The lysates were clarified by centrifugation (5 min, 10,000 × g, 4 °C). Kinase assay was performed at 30 °C for 20 min using 20 μl of lysate in 25 μl of kinase buffer (50 mm sodium β-glycerophosphate pH 7.4, 10 mm magnesium acetate, 0.1 mm EDTA, 50 μm ATP) containing 1.5 μCi of [γ-32P]ATP and 3 μg of Hsp25 (nicely provided by Professor Matthias Gaestel) as substrate. The proteins were resolved by electrophoresis in 7.5–20% gradient SDS-polyacrylamide gel. The gels were dried, and kinase activity was analyzed by autoradiography.Western Blot—The cell lysates were electrophoresed on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (HybondTM; Amersham Biosciences) in 25 mm Tris, 40 mm ϵ-aminocaproic acid, and 20% methanol at 1 mA/cm2 for 90 min. The membranes were blocked for 2 h at 4 °C and incubated overnight at 4 °C with 1:500 4E-BP1 rabbit antibodies (Santa Cruz Biotechnologies), 1:1000 phospho-ERK/MAPK (p44/p42), phospho-JNK/SAPK1 (p54/p46), phospho-p38/SAPK2, ERK/MAPK, JNK/SAPK1, or p38/SAPK2 antibodies (New England Biolabs). Immunoreactive proteins were detected according to the enhanced chemiluminescent protocol (ECL Plus; Amersham Biosciences) using 1:5000 horseradish peroxidase-linked anti-rabbit or anti-mouse secondary antibody. The blots were exposed to film (Hyper-film; Amersham Biosciences) for 1–10 min. The signals were quantified using QuantityOne.Cloning of 4e-bp1 Promoter—A sequence of 1020 bp upstream of the ATG of 4e-bp1 gene was cloned in pCDRIVE vector (Qiagen) by PCR using the 5′-CGGGGGTACCCCGCCTCAAACCCCTGGGCTC-3′ sense primer, the 5′-CCGCTCGAGCGGGTCTCCTGTGCGCTGCAC-3′ antisense primer, and the BAC clone RPCI-11–701H6 (CHORI-BACPAC Resources) as matrix. This sequence was fused to the luciferase gene in the pGL2-Basic vector (Promega) using XhoI and KpnI restriction sites. Entire sequence was verified by sequencing the insert with pGL1 and pGL2 primers (Promega).Transfection and Reporter Assays—HeLa cells were plated 24 h before transfection in 24-well plates so that they are 60–70% confluent the day of transfection. Transfection was realized with LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions. pIRES-EGFP was always cotransfected to estimate the level of transfection (fluorescence measure from lysates with the Victor2) and normalize the luciferase activity. This control level varied from 0.73 to 1.22 times. Luciferase reporter reagent (Promega) was used to measure the luciferase activity according to the manufacturer's instructions in a LB96V luminometer (Berthold Technologies).Histamine and HDC Assays—Histamine concentrations in cell lysates were routinely determined by an automated continuous flow fluorometric technique (lower limit of sensitivity, 0.5 ng/ml), as previously described (49Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62986) Google Scholar). Its specificity has been confirmed by radioimmunoassay (Immunotech, Marseille, France). HDC activity was measured by a radiochromatographic technique, as described before (49Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62986) Google Scholar). Briefly, the cell pellets were resuspended in 50 mm ice-cold phosphate buffer and gently sonicated. Aliquots of the HDC-containing suspensions were then incubated in 50 mm phosphate buffer at a final concentration of 10 μm pyridoxal 5′-phosphate and 0.1 μm l-[3H]histidine (specific activity, 50 Ci/mmol). The incubations were stopped by the addition of perchloric acid (final concentration, 0.4 n) containing 0.3 m unlabeled histidine to minimize possible nonspecific decarboxylation of the remaining l-[3H]histidine. To assess the specificity of the assay, each reaction was performed with or without 10–5m α-fluoromethylhistidine, the specific inhibitor of HDC. After centrifugation, the synthesized [3H]histamine was separated from the [3H]histidine by ion exchange chromatography on Amberlite CG-50 columns.RESULTS4E-BP1 Expression Decreases in Response to PMA—Phosphorylation of 4E-BP1 occurs in response to mitogens or growth factors by activation of the phosphatidylinositol 3-kinase and/or MAPK signal transduction pathways and is rapamycin-sensitive (10Liu G. Zhang Y. Bode A.M. Ma W.Y. Dong Z. J. Biol. Chem. 2002; 277: 8810-8816Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 11Herbert T.P. Tee A.R. Proud C.G. J. Biol. Chem. 2002; 277: 11591-11596Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 50Beretta L. Svitkin Y.V. Sonenberg N. J. Virol. 1996; 70: 8993-8996Crossref PubMed Google Scholar). To assess to what extend PMA modifies 4E-BP1 phosphorylation and/or expression, we analyzed by Western blot the expression and phosphorylation of 4E-BP1 in UT7D1 cells treated with or without PMA for 24 h. It should be mentioned that in all experiments UT7D1 cells were placed in fresh culture medium and GM-CSF-starved for 120 min before the addition of the inhibitors. After 30 min, we added fresh GM-CSF alone or GM-CSF and PMA. 4E-BP1 migrates in 12% SDS-polyacrylamide gels as four bands (α, β, γ, and δ; Fig. 1A). The α band is the less phosphorylated form, and the δ band is the highly phosphorylated isoform. According to our personal observations and in line with the literature, the β band represents the 4E-BP1 isoform phosphorylated on Thr37 and Thr46; the γ isoform is phosphorylated on Thr37, Thr46, and Thr70; and the δ isoform is phosphorylated on these three threonines and on Ser65 (11Herbert T.P. Tee A.R. Proud C.G. J. Biol. Chem. 2002; 277: 11591-11596Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 12Gingras A.C. Raught B. Gygi S.P. Niedzwiecka A. Miron M. Burley S.K. Polakiewicz R.D. Wyslouch-Cieszynska A. Aebersold R. Sonenberg N. Genes Dev. 2001; 15: 2852-2864Crossref PubMed Scopus (1167) Google Scholar). When the cells were cultivated with GM-CSF, the four phosphorylated forms of 4E-BP1 were present. In contrast, when the cells were treated with PMA, only the α, β, and γ forms of 4E-BP1 were barely detected. These results not only reflect a change in the phosphorylation of 4E-BP1 but also a decrease in 4E-BP1 protein amount (Fig. 1A). In the presence of rapamycin, an inhibitor of FRAP/mTOR, or LY294002, an inhibitor of phosphatidylinositol 3-kinase, we observed an accumulation of the α less phosphorylated form of 4E-BP1 in the absence or in the presence of PMA (Fig. 1A). These results are in line with the data from literature that show that the Thr37, Thr46, and Thr70 are phosphorylated by the FRAP/mTOR. p38 and ERK can phosphorylate several sites of 4E-BP1, notably the Ser65 (10Liu G. Zhang Y. Bode A.M. Ma W.Y. Dong Z. J. Biol. Chem. 2002; 277: 8810-8816Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 11Herbert T.P. Tee A.R. Proud C.G. J. Biol. Chem. 2002; 277: 11591-11596Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). This regulation did not occur in our system because treatment of the cells with SB203580 or U0126 did not have any effect on the phosphorylation pattern of 4EBP1. 2M. Rolli-Derkinderen, F. Machavoine, and M. Dy, unpublished results. Nevertheless we could not rule out changes that could have occurred at shorter times of treatment. Northern blot analysis showed that neither rapamycin nor LY294002 affected the transcript level of 4E-BP1, whereas the PMA application resulted in a loss of 4E-BP1 transcript (Fig. 1B). We also looked at the effect of PMA on 4E-BP1 in three different cell lines: 11OC1, HEL, and K562. Twenty-four hours of PMA treatment led to an accumulation of the less phosphorylated isoforms of 4E-BP1 in 11OC1 and K562 cells, whereas in the HEL cell line only the most highly phosphorylated form of 4E-BP1 was detectable (Fig. 1C). In all three cell lines PMA decreased 4E-BP1 expression (Fig. 1C).4e-bp1 Transcript Level Decreases, Whereas the Expression of 4e-bp2 Is Not Sensitive to PMA—Time course analysis showed that 4E-BP1 expression transiently decreased after 4h of GM-CSF (47 ± 17%; Fig. 2, A and C) and that the δ phosphorylated form of 4E-BP1 disappeared after 4 h of GM-CSF treatment. The α less phosphorylated form was mainly detected after 24 h of GM-CSF treatment (Fig. 2A). The expression of 4E-BP1 protein decreased after 4 h of PMA and was undetectable after 24 h (32.5 ± 13% and 4 ± 1%; Fig. 2, A and C). Northern blot analysis showed that the transcript level was strictly correlated with the protein expression level, that PMA negatively regulated 4E-BP1 expression at the transcript level (Fig. 2, B and C), and that this effect lasted 48 h (Fig. 2, D and E). Because 4E-BP2 expression was also known to vary during cell differentiation, we analyzed its expression by Northern blot. The transcript of 4e-bp2 was only present at 24 h of culture in the presence or absence of PMA (Fig. 2, D and E). Taken together, these results showed that the two repressors of translation have different ways of regulating.Fig. 2Kinetics of 4E-BP1 and 4e-bp2 expression. GM-CSF-starved (2 h) UT7D1 cells were cultivated in the presence or absence of PMA for different times as indicated. Samples of cell lysates were subjected to SDS-PAGE followed by Western blotting using either anti-4E-BP1 or anti-actin antibodies (loading control) as indicated (A). The means
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