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Changes in Ribosomal Binding Activity of eIF3 Correlate with Increased Translation Rates during Activation of T Lymphocytes

2005; Elsevier BV; Volume: 280; Issue: 31 Linguagem: Inglês

10.1074/jbc.m414129200

ISSN

1083-351X

Autores

Suzanne Miyamoto, Purvi Patel, John W.B. Hershey,

Tópico(s)

RNA regulation and disease

Resumo

The rate of protein synthesis in quiescent peripheral blood T lymphocytes increases dramatically following mitogenic activation. The stimulation of translation is due to an increase in the rate of initiation caused by the regulation of initiation factor activities. Here, we focus on eIF3, a large multiprotein complex that plays a central role in the formation of the 40 S initiation complex. Using sucrose density gradient centrifugation to analyze ribosome complexes, we find that most eIF3 is not bound to 40 S ribosomal subunits in unactivated T lymphocytes but becomes ribosome-bound following activation. Immunoblot analyses of sucrose gradient fractions for individual eIF3 subunits show that the small eIF3j subunit is unassociated with the eIF3 complex in quiescent T lymphocytes, but upon activation joins the other eIF3 subunits and binds 40 S ribosomal subunits. Because eIF3j has been shown to be required for eIF3 binding to 40 S ribosomes in vitro, the results suggest that mitogenic stimulation of T lymphocytes leads to an activation of eIF3j, thereby enabling eIF3 to bind to the larger ribosome-free eIF3 subunit complex, and then to the 40 S ribosomes. The association of eIF3j with the other eIF3 subunits appears to be inhibited by rapamycin, suggesting a mechanism that lies downstream from the mammalian target of rapamycin kinase. This association requires ionomycin together with a phorbol ester, which also suggests that calcium signaling is involved. We conclude that the complex formation of eIF3 and its association with the ribosomes might contribute to increased translation rates during T lymphocyte activation. The rate of protein synthesis in quiescent peripheral blood T lymphocytes increases dramatically following mitogenic activation. The stimulation of translation is due to an increase in the rate of initiation caused by the regulation of initiation factor activities. Here, we focus on eIF3, a large multiprotein complex that plays a central role in the formation of the 40 S initiation complex. Using sucrose density gradient centrifugation to analyze ribosome complexes, we find that most eIF3 is not bound to 40 S ribosomal subunits in unactivated T lymphocytes but becomes ribosome-bound following activation. Immunoblot analyses of sucrose gradient fractions for individual eIF3 subunits show that the small eIF3j subunit is unassociated with the eIF3 complex in quiescent T lymphocytes, but upon activation joins the other eIF3 subunits and binds 40 S ribosomal subunits. Because eIF3j has been shown to be required for eIF3 binding to 40 S ribosomes in vitro, the results suggest that mitogenic stimulation of T lymphocytes leads to an activation of eIF3j, thereby enabling eIF3 to bind to the larger ribosome-free eIF3 subunit complex, and then to the 40 S ribosomes. The association of eIF3j with the other eIF3 subunits appears to be inhibited by rapamycin, suggesting a mechanism that lies downstream from the mammalian target of rapamycin kinase. This association requires ionomycin together with a phorbol ester, which also suggests that calcium signaling is involved. We conclude that the complex formation of eIF3 and its association with the ribosomes might contribute to increased translation rates during T lymphocyte activation. Primary, mature peripheral blood T lymphocytes are in a natural, quiescent (G0) resting state until challenged by an appropriate mitogenic stimulus that prompts a growth and proliferative immune response. This growth and proliferative response is highly dependent on increased synthesis of proteins from pre-existing and newly generated mRNAs and occurs before new ribosomal RNA synthesis (1.Kay J.E. Ahern T. Atkins M. Biochim. Biophys. Acta. 1971; 247: 322-334Crossref PubMed Scopus (58) Google Scholar, 2.Kay J.E. Ahern T. Lindsay V.J. Sampson J. Biochim. Biophys. Acta. 1975; 378: 241-250Crossref PubMed Scopus (36) Google Scholar). The resting T lymphocyte in culture maintains its quiescent state, exhibiting a low protein synthesis rate even in the presence of nutrients and serum growth factors in the culture media. When primary T lymphocytes are treated in culture with mitogenic stimulants, proteins synthesis rates dramatically increase. This overall increase in protein synthesis is due to an increase in the rate of translation (1.Kay J.E. Ahern T. Atkins M. Biochim. Biophys. Acta. 1971; 247: 322-334Crossref PubMed Scopus (58) Google Scholar, 2.Kay J.E. Ahern T. Lindsay V.J. Sampson J. Biochim. Biophys. Acta. 1975; 378: 241-250Crossref PubMed Scopus (36) Google Scholar, 3.Ahern T. Kay J.E. Biochim. Biophys. Acta. 1973; 331: 91-101Crossref PubMed Scopus (25) Google Scholar) caused by an increase in the initiation rate (2.Kay J.E. Ahern T. Lindsay V.J. Sampson J. Biochim. Biophys. Acta. 1975; 378: 241-250Crossref PubMed Scopus (36) Google Scholar, 4.Ahern T. Sampson J. Kay J.E. Nature. 1974; 248: 519-521Crossref PubMed Scopus (41) Google Scholar, 5.Cooper H.L. Braverman R. J. Cell Physiol. 1977; 93: 213-225Crossref PubMed Scopus (31) Google Scholar, 6.Morley S.J. Rau M. Kay J.E. Pain V.M. Biochem. Soc. Trans. 1993; 21: 397SCrossref PubMed Scopus (4) Google Scholar, 7.Jedlicka P. Panniers R. J. Biol. Chem. 1991; 266: 15663-15669Abstract Full Text PDF PubMed Google Scholar); the rate of polypeptide elongation on the ribosome does not appear to change with stimulation (2.Kay J.E. Ahern T. Lindsay V.J. Sampson J. Biochim. Biophys. Acta. 1975; 378: 241-250Crossref PubMed Scopus (36) Google Scholar). It is suggested that the mechanism responsible for increased translation is complex and involves a number of regulated initiation steps (7.Jedlicka P. Panniers R. J. Biol. Chem. 1991; 266: 15663-15669Abstract Full Text PDF PubMed Google Scholar). These include the phosphorylation state of eIF2α 1The abbreviations used are: eIF, eukaryotic initiation factor; PMA, phorbol myristate acetate (or ‘P‘); I, ionomycin; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; 4E-BP1, eIF4E-binding protein 1; I+P, ionomycin plus PMA; mTOR, mammalian target of rapamycin; MOPS, 4-morpholinepropanesulfonic acid; PHA, phytohemagglutinin. 1The abbreviations used are: eIF, eukaryotic initiation factor; PMA, phorbol myristate acetate (or ‘P‘); I, ionomycin; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; 4E-BP1, eIF4E-binding protein 1; I+P, ionomycin plus PMA; mTOR, mammalian target of rapamycin; MOPS, 4-morpholinepropanesulfonic acid; PHA, phytohemagglutinin. (7.Jedlicka P. Panniers R. J. Biol. Chem. 1991; 266: 15663-15669Abstract Full Text PDF PubMed Google Scholar, 8.Boal T.R. Chiorini J.A. Cohen R.B. Miyamoto S. Frederickson R.M. Sonenberg N. Safer B. Biochim. Biophys. Acta. 1993; 1176: 257-264Crossref PubMed Scopus (33) Google Scholar) and increased eIF2B exchange activity (7.Jedlicka P. Panniers R. J. Biol. Chem. 1991; 266: 15663-15669Abstract Full Text PDF PubMed Google Scholar, 9.Miyamoto S. Kimball S.R. Safer B. Biochim. Biophys. Acta. 2000; 1494: 28-42Crossref PubMed Scopus (23) Google Scholar), both of which affect the level of the ternary complex (eIF2·GTP·Met-tRNAMeti), an intermediate in the binding of the initiator methionyl-tRNA to the 40 S ribosomal subunit (10.Pain V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (636) Google Scholar, 11.Moldave K. Annu. Rev. Biochem. 1985; 54: 1109-1149Crossref PubMed Scopus (357) Google Scholar, 12.Hershey J.W.B. Merrick W.C. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 33-88Google Scholar). Also examined as potential regulatory mechanisms in lymphocytes are eIF4E and 4E-BP phosphorylation (6.Morley S.J. Rau M. Kay J.E. Pain V.M. Biochem. Soc. Trans. 1993; 21: 397SCrossref PubMed Scopus (4) Google Scholar, 8.Boal T.R. Chiorini J.A. Cohen R.B. Miyamoto S. Frederickson R.M. Sonenberg N. Safer B. Biochim. Biophys. Acta. 1993; 1176: 257-264Crossref PubMed Scopus (33) Google Scholar, 13.Morley S.J. Pain V.M. Biochem. J. 1995; 312: 627-635Crossref PubMed Scopus (41) Google Scholar). Phosphorylation of eIF4E was not found to be regulatory, but phosphorylation of 4E-BP1 and the release of eIF4E appear to play a regulatory role in increased initiation of protein synthesis in lymphocytes. However, as suggested above, regulation at several initiation steps likely is involved. We now examine eIF3, the large multisubunit complex involved in the formation of the 40 S preinitiation complex, as a possible regulatory target during the activation of lymphocytes. eIF3 is the largest of the mammalian initiation factors and is implicated in a number of steps in the initiation pathway. It affects ribosome dissociation/re-association, promotes or stabilizes ternary complex binding to 40 S subunits, helps position mRNA on the 40 S ribosome through its interaction with eIF4G, and may contribute to AUG recognition by affecting eIF5 stimulation of eIF2 GTPase activity (reviewed in Refs. 12.Hershey J.W.B. Merrick W.C. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 33-88Google Scholar and 14.Chaudhuri J. Chowdhury D. Maitra U. J. Biol. Chem. 1999; 274: 17975-17980Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). eIF3 consists of 12 non-identical subunits that range in size from 28 to 170 kDa and are named eIF3a (p170), eIF3b (p116), eIF3c (p110), eIF3d (p66), eIF3e (p48), eIF3f (p47), eIF3g (p44), eIF3h (p40), eIF3i (p36), eIF3j (p35), eIF3k (p28), and eIF3l (p69) (15.Browning K.S. Gallie D.R. Hershey J.W. Hinnebusch A.G. Maitra U. Merrick W.C. Norbury C. Trends Biochem. Sci. 2001; 26: 284Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Characterization of eIF3 in mammalian cells has been hampered by its large size and complexity, and only recently have the cDNAs been cloned and sequenced for all of the subunits (16.Asano K. Vornlocher H.P. Richter-Cook N.J. Merrick W.C. Hinnebusch A.G. Hershey J.W. J. Biol. Chem. 1997; 272: 27042-27052Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The functions of the individual subunits are not yet well established. In the yeast Saccharomyces cerevisiae, eIF3 appears to consist of a core complex of only five subunits (eIF3a, -b, -c, -g, and -i), plus a non-stoichiometric subunit, eIF3j. In mammalian cells, the corresponding homologs also may constitute a “core” complex to which the other mammalian subunits bind and regulate eIF3 activity. Yeast eIF3 interacts with eIF1, eIF2β, and eIF5 to form a multifactor complex (17.Phan L. Schoenfeld L.W. Valasek L. Nielsen K.H. Hinnebusch A.G. EMBO J. 2001; 20: 2954-2965Crossref PubMed Scopus (90) Google Scholar, 18.Asano K. Phan L. Anderson J. Hinnebusch A.G. J. Biol. Chem. 1998; 273: 18573-18585Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Mammalian eIF3 also binds eIF1 and eIF5 (19.Das S. Maiti T. Das K. Maitra U. J. Biol. Chem. 1997; 272: 31712-31718Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 20.Bandyopadhyay A. Maitra U. Nucleic Acids Res. 1999; 27: 1331-1337Crossref PubMed Scopus (29) Google Scholar, 21.Majumdar R. Bandyopadhyay A. Maitra U. J. Biol. Chem. 2003; 278: 6580-6587Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and, in addition, binds eIF4B (22.Methot N. Song M.S. Sonenberg N. Mol. Cell Biol. 1996; 16: 5328-5334Crossref PubMed Scopus (156) Google Scholar) and eIF4G (23.Imataka H. Sonenberg N. Mol. Cell Biol. 1997; 17: 6940-6947Crossref PubMed Scopus (237) Google Scholar). Together with its RNA-binding activity and its ability to bind stably to the 40 S ribosome, mammalian eIF3 may play an organizing role on the surface of the 40 S ribosomal subunit. Several lines of evidence suggest that eIF3 activity is regulated during formation of the 40 S preinitiation complex. A small subunit, eIF3j, is cleaved by caspases in apoptosis (24.Bushell M. Wood W. Clemens M.J. Morley S.J. Eur. J. Biochem. 2000; 267: 1083-1091Crossref PubMed Scopus (77) Google Scholar, 25.Clemens M.J. Bushell M. Jeffrey I.W. Pain V.M. Morley S.J. Cell Death Differ. 2000; 7: 603-615Crossref PubMed Scopus (204) Google Scholar), reducing its ability to promote eIF3 binding to the 40 S subunit (26.Fraser C.S. Lee J.Y. Mayeur G.L. Bushell M. Doudna J.A. Hershey J.W. J. Biol. Chem. 2004; 279: 8946-8956Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Apoptosis also leads to eIF3f phosphorylation by an activated, truncated form of cyclin-dependent kinase 11, thereby inhibiting protein synthesis (27.Shi J. Feng Y. Goulet A.C. Vaillancourt R.R. Sachs N.A. Hershey J.W. Nelson M.A. J. Biol. Chem. 2003; 278: 5062-5071Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). eIF3, along with eIF4E, eIF4G, and small, but not large ribosomal subunits, is sequestered in stress granules as part of the stress response in cells, and these stress granules disassemble, and eIF3 returns to its original subcellular localization during recovery from the stress situation (28.Kedersha N. Chen S. Gilks N. Li W. Miller I.J. Stahl J. Anderson P. Mol. Biol. Cell. 2002; 13: 195-210Crossref PubMed Scopus (420) Google Scholar, 29.Dunand-Sauthier I. Walker C. Wilkinson C. Gordon C. Crane R. Norbury C. Humphrey T. Mol. Biol. Cell. 2002; 13: 1626-1640Crossref PubMed Scopus (52) Google Scholar). The interferon-induced protein P56 binds to eIF3e and inhibits translation in vitro by blocking the interaction of eIF3 with the eIF2·GTP·Met-tRNAMeti ternary complex (30.Hui D.J. Bhasker C.R. Merrick W.C. Sen G.C. J. Biol. Chem. 2003; 278: 39477-39482Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The cellular level of eIF3a is reported to vary, with low levels negatively affecting the translation of ribonucleotide reductase M2 mRNA (31.Dong Z. Liu L.H. Han B. Pincheira R. Zhang J.T. Oncogene. 2004; 23: 3790-3801Crossref PubMed Scopus (93) Google Scholar). Finally, numerous eIF3 subunits are implicated in cancer (32.Hershey J.W.B. Miyamoto S. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 637-654Google Scholar), suggesting that regulation of eIF3 activity may be important in cell growth control. Because peripheral blood lymphocytes are in a natural quiescent state, with low protein synthesis (translation) rates, they are an excellent system to study the regulation of translation. It is sometimes difficult to perform growth and proliferation studies in already proliferating, often transformed cell lines because: 1) the normal regulatory pathways may be mutated; 2) the cells need to be synchronized with drugs; and/or 3) the cells need to be serum-starved to reduce translation rates and then re-stimulated with serum or growth factors, leading to low translation rate (2- to 3-fold) increases. Frequent problems are high basal translation rates of serum-deprived cells and/or failure to recover when re-fed with serum. Two signals are required for optimum activation of T lymphocytes in nature. The interaction of the T cell receptor with a major histocompatibility complex-peptide presented by an accessory cell such as the macrophage provides the specific antigen-restricted signal. The second signal is provided through interaction of the CD28 molecule with its cognate ligand, the B7 family of proteins. This second antigen-unrestricted signal provides a co-stimulatory signal necessary for full T cell activation. TCR engagement with the major histocompatibility complex-peptide without B7 co-stimulation results in T cell clonal anergy (33.Lenschow D.J. Walunas T.L. Bluestone J.A. Annu. Rev. Immunol. 1996; 14: 233-258Crossref PubMed Scopus (2327) Google Scholar). Optimal activation of T lymphocytes can be mimicked in cell culture with anti-CD3 and anti-CD28 and results in a sustained increase in protein synthesis rate (34.Mao X. Green J.M. Safer B. Lindsten T. Frederickson R.M. Miyamoto S. Sonenberg N. Thompson C.B. J. Biol. Chem. 1992; 267: 20444-20450Abstract Full Text PDF PubMed Google Scholar). The combination of ionomycin (I) and the phorbol ester, phorbol myristate acetate (PMA or P), also causes a rapid rise in protein synthesis rates and the onset of cell proliferation (4.Ahern T. Sampson J. Kay J.E. Nature. 1974; 248: 519-521Crossref PubMed Scopus (41) Google Scholar, 9.Miyamoto S. Kimball S.R. Safer B. Biochim. Biophys. Acta. 2000; 1494: 28-42Crossref PubMed Scopus (23) Google Scholar, 35.Jagus R. Kay J.E. Eur. J. Biochem. 1979; 100: 503-510Crossref PubMed Scopus (17) Google Scholar), comparable to anti-CD3/28. In contrast, PMA by itself (PMA alone) results in less induction of protein synthesis and no proliferation (9.Miyamoto S. Kimball S.R. Safer B. Biochim. Biophys. Acta. 2000; 1494: 28-42Crossref PubMed Scopus (23) Google Scholar), suggesting that in ionomycin plus PMA (I+P)-activated T lymphocytes the second signal, provided by ionomycin, likely replaces the need for the anti-CD28 co-stimulation. Prior studies in lymphocytes identified signaling pathways that promote the overall increase in global translation rates after activation of T lymphocytes with I+P (9.Miyamoto S. Kimball S.R. Safer B. Biochim. Biophys. Acta. 2000; 1494: 28-42Crossref PubMed Scopus (23) Google Scholar). Translation rates induced by I+P are greater than those induced by PMA alone, suggesting that, additional signals, likely Ca2+-activated, are responsible for the continued rate increase. Translation rates are more sensitive to treatment with PI3K and mTOR inhibitors than to MAPK inhibitors, suggesting that the PI3K and mTOR pathways are more crucial for the translation rate increase (9.Miyamoto S. Kimball S.R. Safer B. Biochim. Biophys. Acta. 2000; 1494: 28-42Crossref PubMed Scopus (23) Google Scholar). Currently, the only translation initiation factors affected by the PI3K or mTOR inhibitors rapamycin and wortmannin are eIF4B (36.Raught B. Peiretti F. Gingras A.C. Livingstone M. Shahbazian D. Mayeur G.L. Polakiewicz R.D. Sonenberg N. Hershey J.W. EMBO J. 2004; 23: 1761-1769Crossref PubMed Scopus (354) Google Scholar) and eIF4E, the latter through the phosphorylation of 4E-BP1 (6.Morley S.J. Rau M. Kay J.E. Pain V.M. Biochem. Soc. Trans. 1993; 21: 397SCrossref PubMed Scopus (4) Google Scholar, 9.Miyamoto S. Kimball S.R. Safer B. Biochim. Biophys. Acta. 2000; 1494: 28-42Crossref PubMed Scopus (23) Google Scholar, 13.Morley S.J. Pain V.M. Biochem. J. 1995; 312: 627-635Crossref PubMed Scopus (41) Google Scholar). However, translation rates are sensitive to the combination of rapamycin and wortmannin, suggesting that these agents not only inhibit 4E-BP1 phosphorylation but also might independently target other translation initiation events. We already know that eIF4E phosphorylation and eIF2B exchange activity are not inhibited by these agents (9.Miyamoto S. Kimball S.R. Safer B. Biochim. Biophys. Acta. 2000; 1494: 28-42Crossref PubMed Scopus (23) Google Scholar), and we cannot rule out a currently “undescribed” activity downstream of mTOR that might affect translation in T lymphocytes. The objectives of this study are to investigate if eIF3 subunit expression and eIF3 activity change in I+P- or PMA-activated T lymphocytes, elucidate how eIF3 interacts with other translational components to form the 40 S preinitiation complex, and determine if eIF3-associated translation initiation events are affected by rapamycin or wortmannin. We report the novel observation that most eIF3 in the unactivated lymphocyte is not associated with 40 S ribosomal subunits, but becomes almost completely associated after 24 h with activation conditions that promote proliferation (I+P) and not with conditions that do not (PMA alone). We also find that eIF3j is not associated with the eIF3 complex but joins the complex following activation. Because eIF3j is required for eIF3 binding to 40 S ribosomes in vitro (26.Fraser C.S. Lee J.Y. Mayeur G.L. Bushell M. Doudna J.A. Hershey J.W. J. Biol. Chem. 2004; 279: 8946-8956Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), the results suggest that eIF3j association with eIF3 is regulated early in the activation response. Thus generation of a complete eIF3 complex, after activation, leads to eIF3–40 S ribosome binding and 40 S preinitiation complex formation, which are events inhibited by rapamycin and wortmannin. Cell culture reagents were obtained from Fisher; fetal bovine serum from Gemini; ionomycin and PMA from Sigma; nylon wool was from Robbins Scientific; goat anti-human eIF3 antibodies and cDNAs to eIF3 subunits have been described previously (16.Asano K. Vornlocher H.P. Richter-Cook N.J. Merrick W.C. Hinnebusch A.G. Hershey J.W. J. Biol. Chem. 1997; 272: 27042-27052Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 37.Meyer L.J. Brown-Luedi M.L. Corbett S. Tolan D.R. Hershey J.W. J. Biol. Chem. 1981; 256: 351-356Abstract Full Text PDF PubMed Google Scholar, 38.Chaudhuri J. 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Preparation and Activation of T Lymphocytes—Human T lymphocytes were obtained from normal human peripheral blood from whole blood buffy coats, discarded tonsils, or leukocyte filters and further enriched for T lymphocytes by nylon wool purification (44.Miyamoto S. Chiorini J.A. Urcelay E. Safer B. Biochem. J. 1996; 315: 791-798Crossref PubMed Scopus (55) Google Scholar). Pig T lymphocytes were isolated from pig whole blood, obtained from the UC Davis Meat Laboratory. Pig blood was mixed with 10% (final concentration) citrate-acetate to prevent coagulation and 1% dextran to help settle red blood cell. The upper layer above the settled red blood cells, containing the white blood cells, was removed and subjected to Ficoll-Hypaque separation (lymphocyte separation media from Cellgro). The buffy coat layer, containing a mixture of T and B lymphocytes, granulocytes, and monocytes, was removed, and the cell mixture was passed through sterile nylon-wool columns to enrich for the T lymphocyte population as previously described (9.Miyamoto S. Kimball S.R. Safer B. Biochim. Biophys. Acta. 2000; 1494: 28-42Crossref PubMed Scopus (23) Google Scholar). Cells were grown in RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 units/ml penicillin/streptomycin, and 100 μg/ml glutamine, at 37 °C, 5% CO2. T lymphocytes were activated with either 0.25 μm ionomycin plus 10 ng/ml PMA (I+P), or 10 ng/ml PMA (PMA alone). For some experiments, T lymphocytes were isolated from human tonsils using these same methods and were found to respond in a similar manner as peripheral blood lymphocytes to I+P or PMA (45.Carini C. Pini C. DiFelice G. Fattorossi A. Fratazzi C. Int. Arch. Allergy Immunol. 1993; 101: 31-38Crossref PubMed Scopus (12) Google Scholar). Immunoblot or Immunoprecipitation of eIF3 and eIF3 Subunits— Whole cell extracts were prepared from T lymphocytes, activated with I+P or PMA (see above), and lysed in SDS-PAGE loading buffer or lysed in 0.4% Nonidet P-40 in Tris buffer containing protease inhibitors. Proteins were resolved by 10% SDS-PAGE and transferred to an Immobilon (Millipore) membrane. The blots were blocked with 1% gelatin (fish, Sigma) in 10 mm Tris-HCl, 0.15 m NaCl with 0.05% Tween 20, probed with anti-human eIF3 goat antibodies and rabbit anti-goat antibodies conjugated with horseradish peroxidase, and developed with chemiluminescent substrate (Lumiglo, Cell Signaling or Western Lightening, PerkinElmer Life Sciences). Immunoblots were exposed to Kodak X-Omat AR or BioMax film. Immunoprecipitations were performed by first absorbing the whole cell extracts with Protein G-Sepharose (Amersham Biosciences), then the lymphocyte whole cell extracts were incubated with 0.5 μl of eIF3 polyclonal antibody (goat) or 1 μl of eIF3a monoclonal antibody, incubated for 1 h, 4 °C in lysis buffer (see above), then 20 μl of washed Protein G-Sepharose was added, incubated 1 h, 4 °C with rotation. The immunoprecipitates were washed (wash, 0.05 m Tris, pH 8, 0.5% Nonidet P-40, 0.14 m NaCl), analyzed by 10% SDS-PAGE, followed by immunoblotting with affinity-purified eIF3j (goat) or eIF3 polyclonal antibody (goat), with ECL (PerkinElmer Life Sciences). Recombinant FLAG-tagged eIF3j was prepared and purified from Escherichia coli. Northern Blot Analysis—Total RNA was isolated from pig lymphocytes (1.54 × 108 cells), unactivated or activated with I+P or PMA alone (0–24 h, TriReagent, Molecular Research Center). RNA concentration was quantitated (A260/280 nm, Beckman DU640), and 10 μg of RNA of each time point was sample separated on a 0.8% formaldehyde agarose gel in MOPS buffer (44.Miyamoto S. Chiorini J.A. Urcelay E. Safer B. Biochem. J. 1996; 315: 791-798Crossref PubMed Scopus (55) Google Scholar), transferred to Nytran membrane (S&S), and cross-linked using UV irradiation (Stratagene). Labeled cDNA probes were prepared from each eIF3 subunit cDNA (provided by the Hershey laboratory) by random prime labeling (Invitrogen) and [32P]dUTP (Amersham Biosciences) and hybridized to the blots (44.Miyamoto S. Chiorini J.A. Urcelay E. Safer B. Biochem. J. 1996; 315: 791-798Crossref PubMed Scopus (55) Google Scholar). After washing, the blots were exposed to Kodak X-Omat AR film and developed or quantitated using a PhosphorImager (Amersham Biosciences). Sucrose Gradient Fractionation of T Lymphocyte Cell Extracts—T lymphocytes were activated with 0.25 μm ionomycin plus 10 ng/ml PMA or 10 ng/ml PMA alone. After activation, cells were harvested, washed, and lysed with cell lysis buffer (10 mm Tris-HCl, pH 7.4, 10 mm MgCl2, 0.25 mm KCl, 1 mm dithiothreitol, and 1% Triton X-100) by incubation on ice for 10 min, followed by centrifugation for 10 min (14,000 × g) to pellet nuclei, microsomes, and unlysed cells. Supernatants were layered on top of sucrose gradients (5–45% sucrose in 50 mm HEPES, pH 7.9, 100 mm NaCl, 10 mm Mg(OAc)2, and 10 units/ml RNasin, Promega) and centrifuged for 4 h at 38,000 rpm, 4 °C (Beckman Ti-41 rotor). Gradients were analyzed by using an ISCO fractionator with UV254 nm detector, and 0.5-ml fractions were collected and precipitated with 10% trichloroacetic acid. Trichloroacetic acid precipitates were centrifuged (30 min, 4 °C, 14,000 rpm, Eppendorf centrifuge), washed twice with acetone, and dissolved in SDS loading buffer. The precipitated proteins were separated by 10% SDS-PAGE, transferred to Immobilon (Millipore) membranes, and subjected to Western blot analysis. The rate of protein synthesis begins to increase in primary peripheral blood lymphocytes grown in culture about 2–3 h after the addition of a combination of ionomycin and PMA (I+P) (9.Miyamoto S. Kimball S.R. Safer B. Biochim. Biophys. Acta. 2000; 1494: 28-42Crossref PubMed Scopus (23) Google Scholar, 46.Grolleau A. Kaplan M.J. Hanash S.M. Beretta L. Richardson B. J. Clin. Invest. 2000; 106: 1561-1568Crossref PubMed Scopus (46) Google Scholar). Translation rates continue to increase in the presence of I+P (8.Boal T.R. Chiorini J.A. Cohen R.B. Miyamoto S. Frederickson R.M. Sonenberg N. Safer B. Biochim. Biophys. Acta. 1993; 1176: 257-264Crossref PubMed Scopus (33) Google Scholar) in a manner similar to stimulation of lymphocytes with phytohemagglutinin (PHA) (47.Kay J.E. Eur. J. Biochem. 1968; 4: 225-232Crossref PubMed Scopus (40) Google Scholar, 48.Kay J.E. Exp. Cell Res. 1969; 58: 185-188Crossref PubMed Scopus (40) Google Scholar) or a combination of anti-CD3 and anti-CD28 (34.Mao X. Green J.M. Safer B. Lindsten T. Frederickson R.M. Miyamoto S. Sonenberg N. Thompson C.B. J. Biol. Chem. 1992; 267: 20444-20450Abstract Full Text PDF PubMed Google Scholar). In contrast, T lymphocytes in the presence of PMA alone (PMA) increase the protein synthesis rate for only the first 8 h, but cannot sustain this rate increase without the presence of the calcium ionophore ionomycin (9.Miyamoto S. Kimball S.R. Safer B. Biochim. Biophys. Acta. 2000; 1494: 28-42Crossref PubMed Scopus (23) Google Scholar). 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