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4E-BP3, a New Member of the Eukaryotic Initiation Factor 4E-binding Protein Family

1998; Elsevier BV; Volume: 273; Issue: 22 Linguagem: Inglês

10.1074/jbc.273.22.14002

ISSN

1083-351X

Autores

Francis Poulin, Anne‐Claude Gingras, Henrik S. Olsen, Simone Chevalier, Nahum Sonenberg,

Tópico(s)

CRISPR and Genetic Engineering

Resumo

Translation initiation in eukaryotes is mediated by the cap structure (m7GpppN, where N is any nucleotide) present at the 5′ end of all cellular mRNAs, except organellar. The cap is recognized by eukaryotic initiation factor 4F (eIF4F), which consists of three polypeptides, including eIF4E, the cap-binding protein subunit. The interaction of the cap with eIF4E facilitates the binding of the ribosome to the mRNA. eIF4E activity is regulated in part by two translational repressors, 4E-BP1 and 4E-BP2, which bind to it and prevent its assembly into eIF4F. We report here the isolation of 4E-BP3, a new member of the 4E-BP family. 4E-BP3 is homologous to 4E-BP1 and 4E-BP2, exhibiting 57 and 59% identity, respectively. The homology is most striking in the middle region of the protein, which contains the eIF4E binding motif and residues that are phosphorylated in 4E-BP1. 4E-BP3 is a heat stable protein that binds to eIF4E in vitro as well as in vivo. Further, 4E-BP3 overexpression specifically reduces eIF4E-dependent translation. The overlapping function and expression of the different 4E-BP family members imply that there is redundancy in this translational control mechanism, underscoring its importance. Translation initiation in eukaryotes is mediated by the cap structure (m7GpppN, where N is any nucleotide) present at the 5′ end of all cellular mRNAs, except organellar. The cap is recognized by eukaryotic initiation factor 4F (eIF4F), which consists of three polypeptides, including eIF4E, the cap-binding protein subunit. The interaction of the cap with eIF4E facilitates the binding of the ribosome to the mRNA. eIF4E activity is regulated in part by two translational repressors, 4E-BP1 and 4E-BP2, which bind to it and prevent its assembly into eIF4F. We report here the isolation of 4E-BP3, a new member of the 4E-BP family. 4E-BP3 is homologous to 4E-BP1 and 4E-BP2, exhibiting 57 and 59% identity, respectively. The homology is most striking in the middle region of the protein, which contains the eIF4E binding motif and residues that are phosphorylated in 4E-BP1. 4E-BP3 is a heat stable protein that binds to eIF4E in vitro as well as in vivo. Further, 4E-BP3 overexpression specifically reduces eIF4E-dependent translation. The overlapping function and expression of the different 4E-BP family members imply that there is redundancy in this translational control mechanism, underscoring its importance. Modulation of gene expression in cells is partly achieved by regulating translation rates. Translation initiation is the major target of such regulation in eukaryotic cells (1Mathews M.B. Sonenberg N. Hershey J.W.B. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 1-30Google Scholar). The rate-limiting step in translation initiation is the binding of the 43 S preinitiation complex to the mRNA. This process is facilitated by the presence of the mRNA 5′-cap structure (2Shatkin A.J. Cell. 1985; 40: 223-224Abstract Full Text PDF PubMed Scopus (193) Google Scholar). Ribosome binding is mediated by eIF4F 1The abbreviations used are: eIF, eukaryotic initiation factor; 4E-BP, eIF4E-binding protein; EST, expressed sequence tag; RLUC, R. reniformis luciferase; FLUC, firefly luciferase; IRES, internal ribosome entry site; POLIRES, poliovirus internal ribosome entry site; GST, glutathioneS-transferase; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; UTR, untranslated region; bp, base pair(s). (3Merrick W.C. Hershey J.W.B. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 31-70Google Scholar, 4Sonenberg N. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 245-270Google Scholar), which consists of three subunits: eIF4E, eIF4A, and eIF4G. eIF4F, in association with eIF4B, is thought to unwind the mRNA secondary structure in the vicinity of the cap to allow the binding of the 43 S preinitiation complex (reviewed in Refs. 4Sonenberg N. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 245-270Google Scholar and 5Pain V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (641) Google Scholar). eIF4E is present in rate-limiting amounts in most cells (6Duncan R. Milburn S.C. Hershey J.W. J. Biol. Chem. 1987; 262: 380-388Abstract Full Text PDF PubMed Google Scholar, 7Hiremath L.S. Webb N.R. Rhoads R.E. J. Biol. Chem. 1985; 260: 7843-7849Abstract Full Text PDF PubMed Google Scholar) (but see Rau et al. (8Rau M. Ohlmann T. Morley S.J. Pain V.M. J Biol Chem. 1996; 271: 8983-8990Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) for an exception in rabbit reticulocytes) and plays a central role in translational control (4Sonenberg N. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 245-270Google Scholar, 5Pain V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (641) Google Scholar). The key role of eIF4E regulation in protein synthesis is underscored by the presence of two related eIF4E-binding proteins, 4E-BP1 and 4E-BP2 (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar, 10Lin T.A. Kong X. Haystead T.A. Pause A. Belsham G. Sonenberg N. Lawrence Jr., J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (613) Google Scholar). These proteins specifically inhibit eIF4E-dependent translation initiation (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar) by competing with eIF4G for binding to eIF4E (11Haghighat A. Mader S. Pause A. Sonenberg N. EMBO J. 1995; 14: 5701-5709Crossref PubMed Scopus (536) Google Scholar). Consequently, 4E-BPs prevent eIF4E association with eIF4G to form the eIF4F complex, resulting in the subsequent inhibition of the 43 S preinitiation complex binding to the mRNA. The competition between 4E-BPs and eIF4G is explained by the presence of a common eIF4E binding site in 4E-BPs and eIF4G (12Mader S. Lee H. Pause A. Sonenberg N. Mol. Cell. Biol. 1995; 15: 4990-4997Crossref PubMed Google Scholar). The binding of 4E-BP1 and 4E-BP2 to eIF4E is controlled by their phosphorylation state. The underphosphorylated forms of 4E-BPs interact with eIF4E, whereas the hyperphosphorylated forms do not (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar, 10Lin T.A. Kong X. Haystead T.A. Pause A. Belsham G. Sonenberg N. Lawrence Jr., J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (613) Google Scholar, 13Gingras A.-C. Sonenberg N. Virology. 1997; 237: 182-186Crossref PubMed Scopus (46) Google Scholar). Upon cell stimulation with serum, growth factors, or hormones, 4E-BP1 becomes hyperphosphorylated and dissociates from eIF4E to relieve the translational inhibition (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar, 10Lin T.A. Kong X. Haystead T.A. Pause A. Belsham G. Sonenberg N. Lawrence Jr., J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (613) Google Scholar, 14Fleurent M. Gingras A.-C. Sonenberg N. Meloche S. J. Biol. Chem. 1997; 272: 4006-4012Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The regulation of eIF4E activity by 4E-BPs is of particular significance considering the transforming potential of eIF4E (15Lazaris-Karatzas A. Montine K.S. Sonenberg N. Nature. 1990; 345: 544-547Crossref PubMed Scopus (861) Google Scholar). Indeed, the overexpression of 4E-BP1 and 4E-BP2 in cells transformed by eIF4E or v-src resulted in a significant reversion of the transformed phenotype (16Rousseau D. Gingras A.-C. Pause A. Sonenberg N. Oncogene. 1996; 13: 2415-2420PubMed Google Scholar). Here we present the molecular cloning and the characterization of 4E-BP3, a new member of the 4E-BP family of translational inhibitors. The cDNA clone no. 87895 (Human Genome Sciences Inc.) was obtained from a prostate carcinoma cell line (LNCaP) EST cDNA library as described previously (17Adams, M. D., Kerlavage, A. R., Fleischmann, R. D., Fuldner, R. A., Bult, C. J., Lee, N. H., Kirkness, E. F., Weinstock, K. G., Gocayne, J. D., White, O.,et al. (1995) Nature377, (suppl.) 3–174Google Scholar). Sequence alignment of 4E-BPs was performed using CLUSTAL W multiple sequence alignment program (version 1.7) (18Thompson J. Higgins D. Gibson T. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56757) Google Scholar). Human 4E-BP3 mRNA distribution was analyzed by Northern hybridization using commercial blots (multiple tissue Northern blot 1 and 2,CLONTECH), with nucleotides 1–597 of the cDNA clone used as a probe, according to the manufacturer's instructions. Anti-4E-BP3 antibody no. 1791 was raised in a rabbit against a synthetic peptide (CVTTPPTAPLSKLEE) comprising amino acids 67–80 of 4E-BP3. The peptide (2 mg) was cross-linked to keyhole limpet hemocyanin using a commercial kit (Pierce) according to the manufacturer's instructions. Anti-4E-BP3 antibody no. 1862 was raised in a rabbit against a GST-4E-BP3 fusion. Anti-eIF4E antibody no. 5853 was described previously (19Frederickson R.M. Montine K.S. Sonenberg N. Mol. Cell. Biol. 1991; 11: 900-2896Crossref Scopus (86) Google Scholar). pcDNA3-4E-BP3 was generated by cloning the coding sequence of 4E-BP3 into pcDNA3 (Invitrogen) using a polymerase chain reaction. Mutants of 4E-BP3 (Y40A or L45A) were generated by polymerase chain reaction mutagenesis and subcloned into pcDNA3. For N-terminally HA-tagged constructs, the coding sequence of 4E-BP3, 4E-BP3-Y40A, or 4E-BP3-L45A was subcloned into pcDNA3-HA (20Imataka H. Olsen H.S. Sonenberg N. EMBO J. 1997; 16: 817-825Crossref PubMed Scopus (175) Google Scholar). pcDNA3-FLAG-eIF4E was constructed by cloning the entire coding sequence of mouse eIF4E into pcDNA3-FLAG using a polymerase chain reaction. The bicistronic reporter plasmid pcDNA3-RLUC-POLIRES-FLUC was generated by subcloning the coding sequence for firefly luciferase (FLUC) from pGEM-LUC (Promega Biotech Inc.), the coding sequence for Renilla reniformis luciferase (RLUC) from pRL-CMV (Promega) and the complete poliovirus type II Lansing 5′-untranslated region (UTR) (poliovirus internal ribosome entry site, POLIRES) (nucleotides 1–737) (21La Monica N. Meriam C. Racaniello V.R. J. Virol. 1986; 57: 515-525Crossref PubMed Google Scholar) into pcDNA3. Polypeptides were resolved on SDS-15% polyacrylamide gels and transferred onto a 0.22-μm nitrocellulose membrane. The membrane was blocked for 16 h at 4 °C with 5% milk in Tris-buffered saline containing 0.5% Tween 20 (TBST). The membrane was incubated for 2 h with primary antibody 1862 (1:1000 in TBST with 1% bovine serum albumin) or 1791 (1:750 in TBST with 1% bovine serum albumin). Incubation with secondary antibody was performed with peroxidase-coupled donkey anti-rabbit Ig (Amersham Corp.) (1:5000 in TBST). Detection was performed with ECL (Amersham Corp.). For adsorption experiments, antiserum (10 μl) was preincubated on ice for 20 min with GST-heart muscle kinase tag (20 μg) or GST-4E-BP3 (20 μg) for antiserum 1862 or with the cognate peptide (20 μg) for antiserum 1791. Capped RNA was synthesized with T7 RNA polymerase in the presence of the cap analog m7GpppG (22Pelletier J. Sonenberg N. Cell. 1985; 40: 515-526Abstract Full Text PDF PubMed Scopus (441) Google Scholar). Wheat germ extract (Promega) (25 μl) was programmed with mRNA in the presence of [35S]cysteine or methionine (25 μCi) according to the manufacturer's instructions. Following translation, extracts programmed with FLAG-eIF4E or 4E-BP3 mRNA were mixed and incubated on ice for 1 h to allow for protein interaction. Protein complexes were recovered at 4 °C for 60 min with 20 μl of m7GDP-agarose resin (23Edery I. Altmann M. Sonenberg N. Gene ( Amst .). 1988; 74: 517-525Crossref PubMed Scopus (87) Google Scholar) previously washed in buffer A (50 mm Tris-HCl, pH 7.5, 150 mm KCl, 1 mm dithiothreitol, 1 mmEDTA, 1 mm phenylmethylsulfonyl fluoride). The resin was washed three times with 1 ml of buffer A, resuspended in Laemmli (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (218068) Google Scholar) sample buffer, and boiled, and proteins were resolved by SDS-PAGE. Gels were processed for fluorography with En3Hance (DuPont). HeLa cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were infected for 1 h in serum-free medium with recombinant vaccinia virus vTF7-3 (25Fuerst T.R. Niles E.G. Studier F.W. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8122-8126Crossref PubMed Scopus (2061) Google Scholar, 26Belsham G.J. Brangwyn J.K. J. Virol. 1990; 64: 5389-5395Crossref PubMed Google Scholar). Cells were then transiently transfected with pcDNA3-RLUC-POLIRES-FLUC (0.75 μg) and either pcDNA3, pcDNA3-4E-BP3, pcDNA3-4E-BP3-Y40A, or pcDNA3-4E-BP3-L45A (2 μg) using Lipofectin (Life Technologies, Inc.) (3.5 μl) as described previously (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar). Cell extracts were prepared in Passive lysis buffer (Promega) 17 h after infection and assayed for RLUC and FLUC activity in a luminometer (BIOORBIT) using a dual-luciferase reporter assay system (Promega). HeLa cells (6-cm dish) were transiently transfected with pcDNA3, pcDNA3-4E-BP3 (6 μg), or with pcDNA3-FLAG-eIF4E (3 μg) and pcDNA3-HA-La, pcDNA3-HA-4E-BP3, pcDNA3-HA-4E-BP3-Y40A, or pcDNA3-HA-4E-BP3-L45A (3 μg) using LipofectAMINE (Life Technologies, Inc.) (10 μl) according to the manufacturer's instructions. Cell extracts were prepared after 24 h by scraping cells in cold buffer A and subjecting the suspension to three freeze-thaw cycles. Cell debris was pelleted by centrifugation, and the protein concentration in the supernatant was determined by the Bio-Rad assay. LNCaP cells were grown in RPMI 1640 containing 10% fetal bovine serum. Cell extracts were prepared in buffer A as described above. LNCaP cell extract (5 mg/ml) was boiled for 8 min and then incubated on ice for 10 min. The precipitated material was removed by centrifugation, and Laemmli sample buffer was added to the supernatant before boiling for 5 min. For co-immunoprecipitation experiments, HeLa cells (6-cm dish) were lysed 24 h after transfection in 500 μl of cold Nonidet P-40 buffer (50 mm Tris-HCl, pH 7.5, 100 mm KCl, 0.5% Nonidet P-40, 1 mmdithiothreitol, 0.5 mm EDTA, 1 mmphenylmethylsulfonyl fluoride), and debris was spun down. Extracts were immunoprecipitated with anti-HA antibody HA.11 (BAbCO) (1.25 μg) for 1 h at 4 °C. Protein G-Sepharose (10 μl) was added, and the mixture was incubated for 1 h at 4 °C. After washing three times with 1 ml of Nonidet P-40 buffer, immunoprecipitates were eluted in Laemmli sample buffer, and proteins were subjected to SDS-PAGE. For m7GDP-agarose precipitation, 1.5 mg of LNCaP cell extract, prepared in buffer A as described above, was incubated with 30 μl of m7GDP-agarose resin for 90 min at 4 °C. The resin was washed three times with 1 ml of buffer A and resuspended in Laemmli sample buffer, and proteins were subjected to SDS-PAGE. LNCaP cells or transfected (pcDNA3 or pcDNA3-4E-BP3) HeLa cells (10-cm dish) were incubated for 5 h at 37 °C in serum-free Eagle's minimal essential medium containing 0.5 mCi/ml [32P]orthophosphate (NEN Life Science Products; 3000 mCi/mmol). The medium was removed, and the cells were rinsed twice with phosphate-buffered saline. Cells were lysed in lysis buffer (10% glycerol, 50 mm Tris (pH 7.5), 60 mm KCl, 2 mm trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 1% Triton X-100, 2 mm dithiothreitol, 50 mm β-glycerolphosphate, 0.1 mm sodium orthovanadate, 1 mm EGTA, 10 mm sodium pyrophosphate, 50 mm sodium fluoride, 1 mmphenylmethylsulfonyl fluoride) for 30 min at 4 °C. Cells were harvested by scraping, and cell debris was removed by centrifugation. The extract was precleared at 4 °C for 1 h by incubation with preimmune serum (7 μl) bound to protein A beads (15 μl). The supernatant was transferred to a fresh tube together with 1862 antiserum (10 μl) bound to protein A beads (25 μl). Incubation end-over-end was carried out for 4 h at 4 °C. Beads were spun down and washed three times with lysis buffer, three times with radioimmune precipitation buffer, and three times with LiCl buffer (500 mm LiCl, 50 mm Tris (pH 7.5)). Immunoprecipitated material was subjected to SDS-PAGE, 15% gel, and transferred to a nitrocellulose membrane, which was dried and autoradiographed. A cDNA for 4E-BP3 (Fig.1 A) was isolated from a LNCaP cDNA library. The cDNA is 698 bp long and consists of a 5′-UTR of 72 bp, an open reading frame of 300 bp, and a 3′-UTR of 326 bp. We do not know if the 5′-UTR of the cDNA is full-length. It is most probably close to full-length, as the size of the mRNA observed in Fig. 3 (800 bp) is close to the size of the cDNA (698 bp), taking into consideration the presence of a poly(A) tail on the mRNA. The 3′-UTR of the cDNA contains a polyadenylation signal (AGUAAA,underlined in Fig. 1 A) located 9 nucleotides upstream of the poly(A) tail. The sequence, although not identical to the consensus polyadenylation sequence (AAUAAA), has been demonstrated to function in some viral cDNAs (27Guntaka R.V. Microbiol. Rev. 1993; 57: 511-521Crossref PubMed Google Scholar) and has not been excluded as a putative polyadenylation signal in eukaryotic mRNAs (28Birnstiel M.L. Busslinger M. Strub K. Cell. 1985; 41: 349-359Abstract Full Text PDF PubMed Scopus (1082) Google Scholar). The open reading frame encodes a predicted 100-amino acid protein with a molecular weight of 10,873. This putative polypeptide is highly homologous to the previously cloned 4E-BP1 and 4E-BP2 proteins, sharing 57 and 59% identity, respectively (Fig. 1 B) (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar). The homology between the 4E-BPs is highest in the middle portion, which contains the eIF4E binding region (12Mader S. Lee H. Pause A. Sonenberg N. Mol. Cell. Biol. 1995; 15: 4990-4997Crossref PubMed Google Scholar) (Fig. 1 B, boxed residues). It is also of interest that all of the phosphorylation sites reported for rat 4E-BP1 (29Fadden P. Haystead T.A. Lawrence Jr., J.C. J. Biol. Chem. 1997; 272: 10240-10247Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) are conserved in 4E-BP2 and 4E-BP3 (Fig. 1 B, denoted by asterisks), with the exception of a conservative change, serine 83 (in 4E-BP1) to a threonine in 4E-BP3. All of the phosphorylation sites contain a (Ser/Thr)-Pro motif.Figure 3Identification of 4E-BP3 in cells. A, an extract (100 μg) from LNCaP cells was resolved by SDS-PAGE, and Western blotting was performed with the following antisera: antiserum 1862 preadsorbed with GST-4E-BP3 (lane 1), antiserum 1862 preadsorbed with GST-HMK (lane 2), antiserum 1791 (lane 3), and antiserum 1791 preadsorbed with its cognate peptide (lane 4). B, an extract (20 μg) from HeLa cells transfected with pcDNA3 (lane 1) or with pcDNA3-4E-BP3 (lane 2) and total (lane 3) or heat-treated (lane 4) extract (250 μg) from LNCaP cells was resolved by SDS-PAGE. Western blotting was performed with antiserum 1791 (1:750).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Data base searches in GenBankTM identified several mouse ESTs that are predicted to encode for a protein highly homologous to the human 4E-BP3. The longest EST (GenBankTM accession no.W18851) is 623 bp long and could encode a protein of 101 amino acids with 86% identity to the human 4E-BP3 (Fig. 1 C). It is therefore most probably the mouse homolog of human 4E-BP3. The tissue distribution of 4E-BP3 mRNA was analyzed by Northern blotting (Fig.2). Two transcripts were detected, a major transcript of approximately 800 bp and a minor transcript of approximately 8.6 kilobase pairs. The smaller transcript most probably corresponds to the cDNA we have cloned here. Although 4E-BP3 RNA is expressed in almost every tissue, the levels of expression vary dramatically among tissues. 4E-BP3 expression is highest in skeletal muscle, heart, kidney, and pancreas, whereas there is very little expression in brain and thymus. The origin of the 8.6-kilobase pair transcript is not clear at present; it could represent a product of alternative splicing or a stable nuclear RNA containing intron sequences. A similar pattern of expression with two different transcripts was also observed for 4E-BP2 (30Tsukiyama-Kohara K. Vidal S.M. Gingras A.-C. Glover T.W. Hanash S.M. Heng H. Sonenberg N. Genomics. 1996; 38: 353-363Crossref PubMed Scopus (49) Google Scholar), possibly pointing to an alternative splicing pattern conserved in 4E-BP3. To determine whether 4E-BP3 protein is expressed in cells, we used two different antisera raised in rabbits. The first (no. 1862) was raised against a GST-4E-BP3 fusion. The second (no. 1791) was raised against a peptide sequence derived from amino acids 67–80 in the C-terminal half of 4E-BP3 (Fig.1 A, bold characters), which is not conserved in the other 4E-BPs (Fig. 1 B). Since 4E-BP3 cDNA was cloned from a cDNA library constructed from an LNCaP human prostate carcinoma cell line (31Webber M.M. Bello D. Quader S. Prostate. 1997; 30: 58-64Crossref PubMed Scopus (158) Google Scholar), an extract from this cell line was used to perform Western blotting. Each of the anti-4E-BP3 antisera detected a single polypeptide of 14.6 kDa (Fig.3 A, lanes 2 and3). Moreover, the band corresponding to the 14.6-kDa polypeptide disappeared when the two antibodies were preincubated with their respective antigens (Fig. 3 A, lanes 1 and4). To further substantiate the authenticity of the 14.6-kDa polypeptide, we expressed 4E-BP3 cDNA in HeLa cells, which do not express 4E-BP3 at detectable levels (Fig. 3 B, lane 1). A polypeptide which co-migrates with 4E-BP3 from LNCaP cells was observed in cells transfected with pcDNA3-4E-BP3 (Fig.3 B, compare lane 2 to lane 3). Since 4E-BP1 and 4E-BP2 are heat stable proteins (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar, 10Lin T.A. Kong X. Haystead T.A. Pause A. Belsham G. Sonenberg N. Lawrence Jr., J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (613) Google Scholar), we wished to determine whether 4E-BP3 is also heat stable. An LNCaP cell extract was boiled, the soluble fraction subjected to SDS-PAGE, and the proteins were transferred to a nitrocellulose membrane. Probing with antisera 1791 demonstrated that 4E-BP3 is heat stable (Fig. 3 B,lane 4). Since the eIF4E binding region in 4E-BPs is highly conserved in 4E-BP3 (Fig. 1 B, boxed residues), it is most likely that 4E-BP3 would also interact with eIF4E. Mutants in the predicted eIF4E binding site were generated to further characterize this interaction. The design of the mutants was based on the results of Mader et al. (12Mader S. Lee H. Pause A. Sonenberg N. Mol. Cell. Biol. 1995; 15: 4990-4997Crossref PubMed Google Scholar) and from analysis of the conserved residues of the eIF4E binding motif found in all of the eIF4E-binding proteins reported so far (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar, 32Gradi A. Imataka H. Svitkin Y.V. Rom E. Raught B. Morino S. Sonenberg N. Mol. Cell. Biol. 1998; 18: 334-342Crossref PubMed Scopus (250) Google Scholar, 33Altmann M. Schmitz N. Berset C. Trachsel H. EMBO J. 1997; 16: 1114-1121Crossref PubMed Scopus (149) Google Scholar, 34Goyer C. Altmann M. Lee H.S. Blanc A. Deshmukh M. Woolford Jr., J.L. Trachsel H. Sonenberg N. Mol. Cell. Biol. 1993; 13: 4860-4874Crossref PubMed Scopus (149) Google Scholar). In the first construct, Tyr-40 of 4E-BP3 was mutated to an alanine. An identical mutation in the eIF4E binding motif of eIF4G abolished its binding to eIF4E (12Mader S. Lee H. Pause A. Sonenberg N. Mol. Cell. Biol. 1995; 15: 4990-4997Crossref PubMed Google Scholar). The second construct was a mutant in which Leu-45 was changed to an alanine. Such a mutation was not analyzed by Maderet al., in which only the two consecutive leucines of the motif were mutated to alanines, resulting in the loss of interaction with eIF4E (12Mader S. Lee H. Pause A. Sonenberg N. Mol. Cell. Biol. 1995; 15: 4990-4997Crossref PubMed Google Scholar). To demonstrate an interaction between eIF4E and 4E-BP3, the proteins were synthesized by in vitro translation and analyzed for complex formation. Presence of the in vitro translated proteins was confirmed by SDS-PAGE, followed by fluorography (Fig.4 A). Very little wild type 4E-BP3 bound to the resin (Fig. 4 B, lane 1). However, a significant amount of 4E-BP3 was retained on the resin in the presence of FLAG-eIF4E (4-fold increase as determined by PhosphorImager analysis, compare lane 2 to lane 1). This demonstrates that, as with 4E-BP1 and 4E-BP2 (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar), association of 4E-BP3 with eIF4E does not affect the binding of eIF4E to the cap structure. Analysis of the binding was also performed using 4E-BP3 containing mutations in the eIF4E binding region. The mutants showed no binding to FLAG-eIF4E (Fig. 4 B, lanes 3and 4). As expected, FLAG-eIF4E bound to the resin (Fig.4 B, lane 5). To confirm the interaction of 4E-BP3 and eIF4E in vivo, co-immunoprecipitations were performed on extracts from HeLa cells transiently transfected with HA-tagged 4E-BP3. The light chain of the anti-HA antibody co-migrates with the endogenous eIF4E on SDS-PAGE, rendering detection of co-immunoprecipitated endogenous eIF4E difficult. To circumvent this problem, FLAG-tagged eIF4E, which migrates more slowly than does endogenous eIF4E, was co-expressed with HA-tagged 4E-BP3, 4E-BP3-Y40A, 4E-BP3-L45A, or La, an unrelated RNA-binding protein (35Chambers J.C. Kenan D. Martin B.J. Keene J.D. J. Biol. Chem. 1988; 263: 18043-18051Abstract Full Text PDF PubMed Google Scholar). Expression of the proteins was confirmed by Western blotting with αHA and αeIF4E (Fig. 4 C). Immunoprecipitations were carried out using a monoclonal anti-HA antibody, and the immunoprecipitates were assayed by Western blotting for the presence of eIF4E and HA-tagged protein. FLAG-eIF4E was not co-immunoprecipitated when cells were co-transfected with the pcDNA3-HA vector (Fig. 4 D, lane 1), nor in the presence of HA-La (Fig. 4 D, lane 2). However, FLAG-eIF4E was co-immunoprecipitated in the presence of HA-4E-BP3 (Fig.4 D, lane 3). 4E-BP3 mutants Y40A and L45A failed to co-immunoprecipitate FLAG-eIF4E (Fig. 4 D, lanes 4 and 5). The expression of the Y40A and L45A constructs was approximately 2–4-fold less efficient as compared with that of the wild type construct (Fig. 4 C, comparelanes 4 and 5 to lane 3), which is expected to affect the amount of FLAG-eIF4E brought down in the assay. However, this cannot explain the complete absence of FLAG-eIF4E. Association of endogenous eIF4E with 4E-BP3 was also examined in LNCaP cells. The presence of eIF4E and 4E-BP3 in LNCaP cell extract was demonstrated by Western blotting with αeIF4E and α4E-BP3 (Fig.4 E, lane 1). eIF4E was precipitated from the extract using m7GDP-agarose resin, and the presence of endogenous eIF4E and 4E-BP3 in the precipitate was observed by Western blotting (Fig. 4 E, lane 2). To determine whether 4E-BP3 inhibits cap-dependent translation, a transient expression assay was used in which HeLa cells were infected with the recombinant vaccinia virus vTF7-3 (25Fuerst T.R. Niles E.G. Studier F.W. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8122-8126Crossref PubMed Scopus (2061) Google Scholar, 26Belsham G.J. Brangwyn J.K. J. Virol. 1990; 64: 5389-5395Crossref PubMed Google Scholar), and co-transfected with 4E-BP3 wild type or mutant constructs and a reporter plasmid. The reporter construct consisted of two cistrons separated by the POLIRES (Fig.5 A). Translation of the RLUC cistron is cap-dependent, whereas that of the FLUC cistron is directed by the poliovirus IRES and is therefore cap-independent (36Pelletier J. Sonenberg N. Nature. 1988; 334: 320-325Crossref PubMed Scopus (1529) Google Scholar). Expression of the 4E-BP3 proteins was assayed by Western blotting, and the results show a similar expression level for all the constructs (Fig. 5 B). The level of expression of RLUC and FLUC was set at 100% when the reporter plasmid was co-transfected with an empty vector (pcDNA3) (Fig. 5, C and D,column 1). When co-transfected with a vector containing the coding sequence for 4E-BP3, the expression of RLUC was decreased by 72% (Fig. 5 C, column 2), whereas the expression of FLUC showed a 2-fold increase (Fig. 5 D, column 2). The Y40A mutant 4E-BP3 as well as the L45A mutant lost their capacity to inhibit cap-dependent translation (Fig.5 C, column 3, 110% of the control; column 4, 90% of the control, respectively). The two mutants did not affect cap-independent translation (Fig. 5 D, columns 3 and 4), in contrast to wild type 4E-BP3. To demonstrate that 4E-BP3 is a phosphoprotein, metabolic labeling of transfected HeLa cells or LNCaP cells was performed using [32P]orthophosphate. Analysis of the immunoprecipitated material shows that 4E-BP3 was labeled with32P in HeLa cells transfected with pcDNA3-4E-BP3 (Fig.6, lane 2), but not in HeLa cells transfected with pcDNA3 (Fig. 6, lane 1). Moreover, the endogenous 4E-BP3 was also found to be labeled in LNCaP cells (Fig. 6, lane 3). Taken together the data indicate that, as with 4E-BP1 and 4E-BP2 (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar, 10Lin T.A. Kong X. Haystead T.A. Pause A. Belsham G. Sonenberg N. Lawrence Jr., J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (613) Google Scholar, 13Gingras A.-C. Sonenberg N. Virology. 1997; 237: 182-186Crossref PubMed Scopus (46) Google Scholar), 4E-BP3 is also a phosphoprotein. We have isolated a new cDNA encoding for a protein that shares a high degree of identity with the translational inhibitors 4E-BP1 and 4E-BP2 (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar) and was thus termed 4E-BP3. Data base searches (NCBI data base and Human Genome Sciences data base) have not revealed any homologies other than to 4E-BP1, 4E-BP2, and to ESTs encoding for the three 4E-BPs. 4E-BP3 was identified by Western blotting as a polypeptide having an apparent molecular mass of 14.6 kDa, which differs from the predicted molecular mass of 10.9 kDa. However, overexpression of 4E-BP3 in HeLa cells and in vitro translation of 4E-BP3 also yielded a polypeptide having an apparent molecular mass of 14.6 kDa, suggesting that the cDNA indeed encodes for a full-length protein. This type of aberrant migration is also observed for 4E-BP1 and 4E-BP2 (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar, 37Hu C. Pang S. Kong X. Velleca M. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3730-3734Crossref PubMed Scopus (130) Google Scholar,38Lin T.A. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 30199-30204Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) and is thought to be due to the high proline content of the proteins, which might affect their mobility in SDS-PAGE (39See Y. Jakowski G. Creighton T.E. Protein Structure: A Practical Approach. IRL Press, Oxford1989: 1-21Google Scholar). Since the 14.6-kDa polypeptide is recognized by two different antibodies and shares the characteristic heat stability of 4E-BP1 and 4E-BP2 (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar, 13Gingras A.-C. Sonenberg N. Virology. 1997; 237: 182-186Crossref PubMed Scopus (46) Google Scholar,38Lin T.A. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 30199-30204Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), it is reasonable to conclude that it is the third member of the 4E-BP family. Interaction of 4E-BP3 with eIF4E was demonstrated by co-immunoprecipitation and by m7GDP-affinity chromatography, both in cell extracts and with in vitrotranslated proteins. The interaction was also analyzed by generating mutants in the eIF4E binding motif of 4E-BP3. The two mutants (Y40A and L45A) did not show any detectable interaction with eIF4E in the assays performed. This result was expected for the Y40A mutant, as it was previously shown that such a mutation in eIF4G abolishes binding to eIF4E (12Mader S. Lee H. Pause A. Sonenberg N. Mol. Cell. Biol. 1995; 15: 4990-4997Crossref PubMed Google Scholar). The inability to detect an interaction of the L45A mutant with eIF4E demonstrates that the first of the two consecutive leucine residues in the eIF4E binding motif, alone dramatically affects the interaction. This result is not surprising as the first leucine is absolutely conserved in all of the eIF4E-binding proteins from yeast to human (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar, 32Gradi A. Imataka H. Svitkin Y.V. Rom E. Raught B. Morino S. Sonenberg N. Mol. Cell. Biol. 1998; 18: 334-342Crossref PubMed Scopus (250) Google Scholar, 33Altmann M. Schmitz N. Berset C. Trachsel H. EMBO J. 1997; 16: 1114-1121Crossref PubMed Scopus (149) Google Scholar, 34Goyer C. Altmann M. Lee H.S. Blanc A. Deshmukh M. Woolford Jr., J.L. Trachsel H. Sonenberg N. Mol. Cell. Biol. 1993; 13: 4860-4874Crossref PubMed Scopus (149) Google Scholar, 40Yan R. Rychlik W. Etchison D. Rhoads R.E. J. Biol. Chem. 1992; 267: 23226-23231Abstract Full Text PDF PubMed Google Scholar). The effect of the Y40A and L45A mutants on translation was also assessed in a transient expression assay in which 4E-BP3 was shown to inhibit eIF4E-dependent translation by 72%. As expected from mutants with an impaired ability to bind to eIF4E, no effect was observed on eIF4E-dependent translation. The mutants also abolished the stimulation (2-fold) of IRES-mediated translation by 4E-BP3. Such a stimulation has not been observed in earlier reports with 4E-BP1 and 4E-BP2 (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar, 41Ohlmann T. Rau M. Pain V.M. Morley S.J. EMBO J. 1996; 15: 1371-1382Crossref PubMed Scopus (182) Google Scholar). The stimulation is specific as the mutants that are defective in binding to eIF4E, although expressed at a level similar to that of wild type 4E-BP3 (Fig. 5 B, comparelanes 3 and 4 to lane 2), lost the ability to stimulate IRES-dependent translation. Since 4E-BPs and eIF4G compete for a limiting amount of eIF4E, it is possible that the high level of overexpression of 4E-BP3 in this system (Fig.5 B, lane 2) releases more of eIF4G that can participate in IRES-mediated translation and thus increase the expression of FLUC. 4E-BP3 mRNA tissue expression differs from that of 4E-BP1 and 4E-BP2 (30Tsukiyama-Kohara K. Vidal S.M. Gingras A.-C. Glover T.W. Hanash S.M. Heng H. Sonenberg N. Genomics. 1996; 38: 353-363Crossref PubMed Scopus (49) Google Scholar, 37Hu C. Pang S. Kong X. Velleca M. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3730-3734Crossref PubMed Scopus (130) Google Scholar). This may suggest that the regulation of eIF4E function by the 4E-BPs is modulated in a tissue-specific manner and is dependent upon the relative abundance of each 4E-BP. Since the 4E-BPs share biochemical and functional characteristics, it is likely that their relative activity in a given tissue or cell type is regulated post-translationally. It is well established that 4E-BP1 activity is regulated via phosphorylation (9Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1079) Google Scholar, 14Fleurent M. Gingras A.-C. Sonenberg N. Meloche S. J. Biol. Chem. 1997; 272: 4006-4012Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 42Gingras A.-C. Svitkin Y. Belsham G.J. Pause A. Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5578-5583Crossref PubMed Scopus (200) Google Scholar, 43von Manteuffel S.R. Gingras A.-C. Ming X.F. Sonenberg N. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4076-4080Crossref PubMed Scopus (224) Google Scholar). Since 4E-BP3 is a phosphoprotein, it is highly likely that its activity is also regulated by its phosphorylation state. A differential regulation of the phosphorylation of the 4E-BPs could explain the redundancy of their function. However, recent reports show that 4E-BP1 and 4E-BP2 behave similarly in response to insulin and cAMP treatment (38Lin T.A. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 30199-30204Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) or to adenovirus infection (13Gingras A.-C. Sonenberg N. Virology. 1997; 237: 182-186Crossref PubMed Scopus (46) Google Scholar, 44Feigenblum D. Schneider R.J. Mol. Cell. Biol. 1996; 16: 5450-5457Crossref PubMed Scopus (67) Google Scholar). Therefore, the overlapping expression of the 4E-BPs may also reflect a safeguard mechanism. In support of such a hypothesis, a mouse knock-out of 4E-BP1 failed to show any phenotype 2K. Tsukiyama-Kohara and N. Sonenberg, unpublished data. (45Blackshear P.J. Stumpo D.J. Carballo E. Lawrence Jr., J.C. J. Biol. Chem. 1997; 272: 31510-31514Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), suggesting that the overlapping expression of other 4E-BPs may compensate for the disrupted 4E-BP1. This underscores the importance of 4E-BPs in regulating eIF4E-dependent translation initiation. In conclusion, the data presented show that 4E-BP3 shares biochemical and functional characteristics with 4E-BP1 and 4E-BP2. To examine whether the 4E-BPs are differentially regulated, it will be important to determine if 4E-BP3 behaves in a manner similar to 4E-BP1 and 4E-BP2 in response to extracellular stimuli. Specifically, analysis of the phosphorylation of the 4E-BPs under various conditions will be required to ascertain if differences among the 4E-BPs exist. We thank Colin Lister for excellent technical assistance, Hiroaki Imataka for pcDNA3-HA and pcDNA3-HA-La, Shigenobu Morino for pcDNA3-FLAG, France Landry for help with some of the experiments, and Christine DeMaria, Mathieu Miron, and Brian Raught for critical reading of the manuscript.

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