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J. Biol. Chem., Vol. 280, Issue 31, 28251-28264, August 5, 2005
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From the
Department of Biochemistry and Molecular Medicine, School of Medicine, University of California-Davis, Davis, California 95616 and the Division of Hematology/Oncology, Cancer Center, University of California-Davis, Sacramento, California 95817
Received for publication, December 15, 2004 , and in revised form, May 24, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is suggested that the mechanism responsible for increased translation is complex and involves a number of regulated initiation steps (7). These include the phosphorylation state of eIF2
1 (7, 8) and increased eIF2B exchange activity (7, 9), 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 (10–12). Also examined as potential regulatory mechanisms in lymphocytes are eIF4E and 4E-BP phosphorylation (6, 8, 13). 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 and 14). 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). 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). 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, eIF2
, and eIF5 to form a multifactor complex (17, 18). Mammalian eIF3 also binds eIF1 and eIF5 (19–21) and, in addition, binds eIF4B (22) and eIF4G (23). 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, 25), reducing its ability to promote eIF3 binding to the 40 S subunit (26). Apoptosis also leads to eIF3f phosphorylation by an activated, truncated form of cyclin-dependent kinase 11, thereby inhibiting protein synthesis (27). 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, 29). 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). The cellular level of eIF3a is reported to vary, with low levels negatively affecting the translation of ribonucleotide reductase M2 mRNA (31). Finally, numerous eIF3 subunits are implicated in cancer (32), 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). 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). 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, 9, 35), comparable to anti-CD3/28. In contrast, PMA by itself (PMA alone) results in less induction of protein synthesis and no proliferation (9), 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). 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). Currently, the only translation initiation factors affected by the PI3K or mTOR inhibitors rapamycin and wortmannin are eIF4B (36) and eIF4E, the latter through the phosphorylation of 4E-BP1 (6, 9, 13). 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), 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), 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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). 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). 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).
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 x 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), 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). 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 x 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Biochemical studies of translational control in lymphocytes have been conducted using human or porcine (pig) lymphocytes (2, 6, 54–56). In early studies, pig lymphocytes were commonly used as an alternate source of primary lymphocytes (2, 54–56). Fresh pig blood can be obtained directly following slaughtering, and T lymphocytes are rapidly prepared in large enough amounts for biochemical studies. Pig T lymphocytes can be activated with PHA, PMA, or the combination of I+P (2, 8, 9, 54–57) in a manner similar to human T lymphocytes (34). An example of the increase in translation rate induced by I+P in pig lymphocytes is shown in Fig. 1. Similar to results from human T lymphocytes, protein synthesis rates in pig T lymphocytes increase 3-fold in the first 8 h of activation by I+P and continue to increase thereafter (data not shown) (2).
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35 kDa is detected in the cell lysates from human and pig lymphocytes, showing the specificity of this antibody for eIF3j. To provide further proof that antibodies in the anti-eIF3 serum recognize pig eIF3j, a human recombinant eIF3j was used to compete with the pig protein for the antibodies. Purified recombinant His-tagged eIF3j migrates slightly more slowly than native eIF3j during SDS-PAGE (Fig. 2C, left, Coomassie stain) and is recognized by anti-eIF3 (Fig. 2C, right, Immunoblot). Jurkat, unactivated, and activated pig and human lymphocyte cell lysates were subjected to immunoprecipitation with anti-eIF3 antiserum. Immunoprecipitates were analyzed by SDS-PAGE and Western blotting with the same eIF3 polyclonal antiserum (Fig. 2D, top panel) and with the affinity-purified eIF3j antibody (Fig. 2D, bottom panel). eIF3a and eIF3b is detected in all samples, although these subunits appear to migrate slightly slower in unactivated pig lymphocytes, suggesting the possibility of post-translation modification of these eIF3 subunits. The eIF3j affinity antibody detected eIF3j in unactivated and activated pig lymphocytes, and in activated human lymphocytes, but was unable to detect it in unactivated human lymphocytes, and it appears to be below the detection limit in the human unactivated lymphocytes, likely due to the smaller number of human lymphocytes (0.5–1 x 108 cells) compared with pig (2 x 108 cells). The difference in cell number is because of the greater availability of pig lymphocytes. Finally, duplicate blots of eIF3 immunoprecipitates from Jurkat, unactivated, and activated pig lymphocyte cell lysates were prepared and incubated with the same eIF3 polyclonal antibody in the absence or presence of purified recombinant eIF3j (Fig. 2E). eIF3j is detected in the Jurkat and activated pig lymphocytes lysates (Fig. 2E, lanes 1 and 3 and part of lane 4, still attached to the first blot), but not detected in the blot incubated with recombinant eIF3j (Fig. 2E, lanes 4–6), nor with purified eIF3 (Fig. 2E, lane 7). The failure to detect eIF3j is not due to a loss of eIF3j protein on the membrane, because stripping and re-probing with antibodies to eIF3j readily detect the protein (Fig. 2E, right side of figure). eIF3j in the immunoprecipitates from unactivated pig lymphocytes is not easily detected by the polyclonal eIF3 antibody, but is weakly detected by the affinity-purified eIF3j antibody (Fig. 2E, right, lane 2, also observed in Fig. 2D). In summary, because anti-eIF3j antibodies in the eIF3 antiserum recognize a pig protein of the same size as human eIF3j, and adding purified recombinant eIF3j reduces this recognition, we conclude that the eIF3j band in pig lymphocytes is truly the j-subunit of pig eIF3.
eIF3 Protein Levels Remain Constant and Then Increase 16–24 h after Stimulation with I+P—To determine if gene expression (protein and mRNA) of eIF3 subunits increases with activation of T lymphocytes and correlates with the increase in translation rate (Fig. 1), we performed Western blot and Northern blot analyses of eIF3 subunits in unactivated and activated lymphocytes. We suspected that eIF3 protein levels might be low and limiting in unactivated lymphocytes, and increased expression might be needed for the increase in translation rate. Therefore we investigated whether eIF3 protein levels change after activation of human or pig T lymphocytes with I+P or PMA alone. Western blot analysis of eIF3 during the first 8 h after I+P activation shows no change in eIF3 protein level in human (Fig. 3A) or pig T lymphocytes (Fig. 3B). A longer I+P activation period (24 h) with pig T lymphocytes shows large increases in eIF3 at 16 and 24 h (Fig. 3C). In contrast, activation of pig T lymphocytes with PMA alone results in no increase in eIF3 protein during the first 4 h of activation and shows only a slight increase at 16 and 24 h (Fig. 3D), suggesting that part of the longer, sustained increase in translation rate induced by I+P activation in lymphocytes may be due to increased eIF3 protein levels promoted by the presence of ionomycin during activation. Because eIF3 levels do not change in the first 8 h, any change in translation due to eIF3 function must be ascribed to increased specific activity and not to increased protein. In these experiments, two detergent methods of cell lysis were used, Nonidet P-40 and SDS lysis, with similar results, although cell extracts from unactivated pig T lymphocytes (Fig. 3C, lane 1) when prepared with Nonidet P-40 detergent appear to show slightly less eIF3 than when prepared with SDS lysis buffer (Fig. 3B, lane 1). eIF3j protein expression during lymphocyte activation was separately examined using the affinity antibody (Fig. 3D, middle panel), and little change in eIF3j or eIF3a protein levels was observed in the 8 h after activation but did increase after 16 and 24 h of activation with I+P. In contrast, -actin protein is highly expressed in unactivated lymphocytes and appears to slightly decrease during the early activation period (2 h). As was observed earlier (57), levels are high in unactivated lymphocytes, may slightly decrease, but actin protein levels appears to remain relatively constant until 24 h of activation.
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Subunits eIF3b, eIF3d, and eIF3i appear to have multiple mRNA forms. eIF3b has an unusual gene expression pattern, with at least four mRNAs; two mRNAs (3.0 and 5.4 kb) appear in unactivated pig T lymphocytes, whereas the other two mRNAs (2.0 and 8.4 kb) appear to be expressed after activation. Interestingly, the largest 8.4-kb mRNA was expressed at 16 and 24 h in T lymphocytes activated by both the I+P and PMA alone, although the transcript was less well expressed in cells activated with PMA alone. Subunit eIF3d exhibited a major 2-kb and a minor 3-kb mRNA, both of which increased during longer activation times. The major eIF3i band was only about 0.24 kb, and therefore was too small to be the mRNA encoding eIF3i and is likely an artifact. The minor eIF3i band at 1.35 kb was therefore the only form of the eIF3i mRNA and was induced by both I+P and PMA alone.
Overall, gene expression of most of the "core" eIF3 subunits (eIF3a, eIF3b, eIF3c, and eIF3i) appeared to be strongly induced by I+P, less so by PMA alone, whereas two (subunits g and j) were barely induced at all, suggesting that they might be coordinately expressed. Importantly, the increases in mRNA levels for most eIF3 subunits occurred before increases in protein levels. This "lag" in synthesis of eIF3 subunit proteins correlates with similar lags already reported for eIF2(34), eIF4E (8), and eIF2B (62). The lag in new eIF synthesis is likely due in large part to the low translation rates in early activated lymphocytes. However, further work is needed to determine whether or not eIF mRNAs are specifically regulated at the translational level in activating T lymphocytes. These observations suggest that transcriptional up-regulation of eIF genes may be important for the later increase in translation rates (after 8 h) in lymphocytes, but not for the initial 8-h increase. Increased eIF3 subunit gene expression also may be important for T lymphocyte proliferation, because I+P and not PMA alone causes the greatest mRNA increase for most eIF3 subunits.
We also measured the amount of total RNA in our T lymphocyte samples, activated with I+P or PMA alone, to determine if an increase in total RNA/cell correlates with the translation rate increases induced by each condition. The results show that total RNA (per cell) did not increase in lymphocytes activated with I+P or with PMA alone for the first 8 h (Fig. 4B). However, after 24-h activation, there was an almost 4-fold increase with I+P, but not with PMA alone. Because ribosomal RNA is the major contributor to total RNA (80%), the constant total RNA levels through 8 h of activation suggest that new ribosomes are not generated, and therefore are not responsible for the increase in protein synthesis activity. However, new ribosome biogenesis at later times, together with new eIF synthesis, certainly contributes to enhancement of translation rates at later times. Apparently, ribosome biogenesis following T lymphocyte activation is dependent on calcium-activated pathways induced through ionomycin. It is important to note that the increases in eIF3 subunit mRNAs shown in Fig. 4A undermeasure the mRNA levels in late activated T lymphocytes, because equal amounts of RNA were examined, not RNA from an equal number of cells.
eIF3 Is Not Bound to 40 S Ribosome Subunit in Quiescent T Lymphocytes—Previous analyses of eIF3 binding to ribosomes in rabbit reticulocyte lysates (63–65) and lysates of cultured transformed cells (26) show that eIF3 binds stably to 40 S ribosomal subunits and is found in the 80 S and polysome regions of the sucrose gradients used to fractionate the cell lysates (63). A minor portion of the eIF3 also is found in the 15 S region of the gradient, corresponding to free, unbound eIF3 (63). The finding that purified eIF3 binds in vitro to 40 S ribosomal subunits in the absence of all other translational components (66) reinforces the view that native 40 S ribosomal subunits contain eIF3. Given that resting primary T lymphocytes are in a "natural" quiescent Go state without the need to manipulate them into a "pseudo" quiescent state, we felt that the true state of eIF3 in Go and its association with ribosomes would be best studied in resting lymphocytes. To address this issue, sucrose gradient centrifugation was performed on lysates isolated from quiescent pig T lymphocytes, T lymphocytes activated with I+P or PMA alone, and exponentially growing human Jurkat T leukemic cells (Fig. 5). Fractions from each sucrose gradient were analyzed by Western immunoblotting as described under "Materials and Methods." Surprisingly, nearly all of the eIF3 in unactivated pig lymphocytes is located at about 15 S between the top of the gradient and the 40 S ribosomal subunit (Fig. 5A, fractions 3–5). The sedimentation position suggests that in unactivated G0 lymphocytes eIF3 exists as a free factor not associated with the 40 S ribosomal subunit. Only a very small amount of eIF3 was detected in fractions corresponding to the 40 S ribosome in the unactivated pig T lymphocytes (Fig. 5A, fractions 9–11). The absence of ribosome-bound eIF3 correlates with the low rate of translation in these cells.
eIF3 Shifts to the 40 S Subunit upon Stimulation with I+P— When pig T lymphocytes are activated for 24 h with I+P and analyzed by sucrose gradient centrifugation, nearly all of the eIF3 shifts from the 15 S to the 40 S region of the gradient where the small ribosomal subunit sediments (Fig. 5B). A similar location for eIF3 was seen when Jurkat cells were analyzed (Fig. 5D). However, an extensive shift was not seen following 24-h activation with PMA (Fig. 5C). Instead, only very small quantities of eIF3 were 40 S ribosome-bound, the vast amount remained as free, unbound eIF3. Thus, the shifting of eIF3 from a free state to one bound to 40 S ribosomal subunits, correlates with the increased translation rate.
The results obtained with the Jurkat T cell line (Fig. 5D) are similar to those obtained with many other cultured cell lines (41). Jurkat T cells are rapidly proliferating lymphocytes with high rates of translation (67). They contain two mutations in the PTEN (phosphatase and tensin homolog) phosphatase gene and are interleukin-2-independent. Results from the Western blot analysis for eIF3 in sucrose gradient fractions of Jurkat T cell extracts show that all of the eIF3 is associated with the 40 S subunit fraction, and none is in the free state (Fig. 5D). A similar result was seen when Jurkat T cells were serum-starved for 24 or 48 h (results not shown). Clearly, eIF3 is fully capable of binding to ribosomes in proliferating Jurkat T cells.
The kinetics of eIF3 association with the 40 S ribosomal subunit was investigated next. A time course of I+P stimulation of pig T lymphocytes was performed followed by sucrose gradient fractionation of cell extracts and SDS-PAGE and Western blot analysis of sucrose gradient fractions. Results are shown only for times 0, 2, and 4 h of stimulation (Fig. 6). eIF3 did not associate with the 40 S subunit (identified by vertical arrows in Fig. 6 near fraction 10) after 2 h of stimulation. However, a distinct shift of eIF3 proteins toward the 40 S region was seen at 4 h, where some of the eIF3 was bound to the 40 S subunit. This increased association correlates with the activation of protein synthesis, where a 2-fold increase was seen at 4 h (Fig. 1).
eIF3 Complex Composition Changes following I+P Activation of T Lymphocytes—A careful evaluation of eIF3 subunits in the Western blots of quiescent pig T lymphocytes shown in Figs. 5 and 6 indicates that eIF3j is not present in, or is substantially dissociated from, the eIF3 complex located at about 15 S in the sucrose gradients. eIF3j is predominantly in fractions 1 and 2 (Figs. 5 and 6) in quiescent cells, but becomes associated with the eIF3 complex and ribosomes upon activation. Two other high molecular weight bands also appear to be unassociated with eIF3, occurring in fractions 1 and 2 (Figs. 5 and 6). However, these bands do not correspond to eIF3a and eIF3b/c; their appearance in these fractions is not reproducible and appears to be an artifact of this experiment.
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The presence of another eIF3 subunit, eIF3f, was also immunoblotted with a specific antibody (27) (Fig. 7B). eIF3f also appears to shift into larger eIF3 complexes with activation of lymphocytes. It appears to form an intermediate complex with the other eIF3 subunits and then a complex that associates with the ribosomes in the activated lymphocytes. It is barely detectable in unactivated lymphocytes and appears to be found in slightly less dense fractions than eIF3a and eIF3b. The reason for this difference is under investigation.
The situation with eIF3j is more intriguing. Our recent in vitro experiments performed with purified eIF3 complexes and eIF3j indicate that eIF3j is required for eIF3 binding to 40 S ribosomal subunits (26). eIF3 complexes lacking only eIF3j bind with low affinity, whereas addition of eIF3j results in tight binding. The data from Figs. 5 and 6 suggest that eIF3j is not part of the eIF3 complex in unactivated pig and human lymphocyte lysates, but rather barely sediments into the gradient. However, after I+P activation, eIF3j is no longer unassociated from eIF3, but appears to become part of the larger eIF3 ribosome-free complex, and is found in gradient regions rich in ribosomes. To confirm these observations in human T lymphocytes, we employed the affinity-purified anti-eIF3j antibody that was characterized in Fig. 2. As shown in Fig. 7B, eIF3j from quiescent human T lymphocytes again is found in gradient fractions 1 and 2, well separated from the eIF3 complex (fractions 5 and 6). This experiment was conducted with human lymphocytes after 10 h of activation, so the data may appear different from the other experiments that were conducted with 24 h of activation. Also, we were unable to detect eIF3j in the intermediate ribosome-free complex, likely due to the low sensitivity of the affinity antibody and the low amount of eIF3j present in these human lymphocyte samples. Similar experiments conducted with larger numbers of pig lymphocytes did show the intermediate form of eIF3 with eIF3j (data not shown). We suspect that, upon activation, eIF3j leaves its unassociated free state, becomes associated with the larger eIF3 complex, and then this complex binds to the small ribosomal subunit.
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The behavior of eIF3j is consistent with the hypothesis that eIF3j is in an inactive state in quiescent cells and thereby contributes to the low level of initiation of protein synthesis. Its ability to form a complex with other eIF3 subunits and bind to 40 S ribosomal subunits appears to be contingent on stimulation of T lymphocytes and may therefore play a key role in the induction of optimal translation rates needed for proliferation. We clearly demonstrate that eIF3j associates with eIF3a in Jurkat T leukemic cells and in activated T lymphocytes, but this association is absent in unactivated T lymphocytes.
The same blots in Fig. 7 (A and B) used for detecting the presence of individual eIF3 subunits in sucrose gradients were stripped and re-probed with specific antibodies to eIF4G, eIF4E, eIF4B, and eIF2. eIF4G, eIF4E, eIF4B, and eIF2 in unactivated lymphocytes are found near the top of the gradient, indicative of their not being a part of either eIF3 or ribosomal complexes (Fig. 7C). eIF4E in unactivated lymphocytes mostly appears in the least dense fractions and seems to be in a different fraction from eIF4G and eIF4B, thereby suggesting that it is not associated with eIF4G or eIF4B in unactivated lymphocytes. Upon activation of lymphocytes (human) with I+P, a portion of each of the three factors sediments somewhat further into the gradient, around fractions 4 and 5, but none appears to bind to 40 S ribosomes. The increase in density/size of eIF4E supports the findings of Morley et al. (13), in lymphocytes. Interestingly, eIF4B shifts to a different fraction from eIF4E and eIF4G. The failure of these initiation factors to bind to ribosomes contrasts with what is seen with eIF3. The absence of a shift of eIF2 to the 40 S region of the gradient may be due to the low level of I+P activation at 10 h. These results are consistent with earlier experiments of Boal et al. (8), who demonstrated that there did not appear to be a change in the association of the ternary complex with ribosomes during the activation of human T lymphocytes.
Treatment of T Cells with Rapamycin Inhibits eIF3 Association with 40 S Ribosomal Subunits—We previously reported that inhibitors of important signaling pathways in lymphocytes also prevent increased translation rates in early I+P-activated lymphocytes (9). Particularly effective at inhibiting the stimulation of protein synthesis are rapamycin, an inhibitor of the mTOR pathway, and wortmannin, an inhibitor of the phosphatidylinositol 3-kinase (PI3K) and Akt pathway. Less effective at preventing increased translation rates are the MAPK inhibitors (9). Therefore, if eIF3 complex formation and association with the 40 S ribosomal subunit are necessary for increased protein synthesis after activation of lymphocytes, agents that inhibit increased translation rates might also inhibit this process. We asked whether or not these protein kinase inhibitors affect eIF3 recruitment to the 40 S ribosomal subunit. Quiescent human T lymphocytes were pre-treated with rapamycin or wortmannin and then activated with I+P for 10 h. Cell extracts were fractionated by sucrose gradient centrifugation and analyzed by Western blotting. As already shown, eIF3 in unactivated cells is mostly in the 15 S region of the gradient, with very little bound to 40 S ribosomal subunits (Fig. 9A), whereas following I+P stimulation, a substantial amount of eIF3 becomes bound to the 40 S subunits (Fig. 9B). When rapamycin (Fig. 9C) or wortmannin (Fig. 9D) are present, the shift of eIF3 onto the 40 S ribosomal subunits is reduced. Therefore, the PI3 kinase pathway operating through mTOR appears to contribute to the stimulation of eIF3 binding to 40 S subunits. It is known that rapamycin or wortmannin treatment prevents the phosphorylation of 4E-BP1 in T lymphocytes activated by I+P, and that this would be expected to prevent translational activation (9). The novel finding reported here indicates that these signal transduction pathways also operate on eIF3–40 S binding, which are events not expected to be affected by the phosphorylation levels of 4E-BPs.
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The finding that numerous subunits of eIF3 are expressed at significantly higher levels during the latter half of the 24 h T cell activation regimen with I+P is not surprising, because a number of other initiation factors have been shown to increase in amounts during this period (7, 8, 34, 62). It is surprising, however, that the eIF3 subunits are not all regulated (transciptionally) identically, because each is present in the eIF3 complex at near-stoichiometric amounts. Only two subunits, namely eIF3f and eIF3j, fail to be induced, but instead appear in quiescent cells at cellular levels similar to those following 24-h activation with I+P. It may be pertinent that eIF3f is regulated in the A375 human melanoma cell line during apoptosis, when a constitutive active form of cyclin-dependent kinase 11 phosphorylates the subunit and converts it into an inhibitor of protein synthesis (27). eIF3j also may be a regulated subunit, as discussed below.
Examination of eIF3 in T lymphocyte whole cell extracts fractionated by sucrose gradient centrifugation led to the unexpected finding that eIF3 is not bound to 40 S ribosomes in quiescent cell lysates, but rather occurs as a free eIF3 complex. eIF3 in proliferating cells is mainly bound to ribosomes, as shown here in Jurkat cells (Fig. 5D) and in numerous earlier studies (63, 68). Furthermore, stable eIF3 binding to 40 S subunits in vitro does not require any other component of initiation (66). It appears that either something is missing or defective in the eIF3 or 40 S ribosomal subunit that is required for binding, or that one or more components in these complexes is modified to prevent a stable association. Interestingly, Kay et al. (69) previously described the presence of an "inhibitor" in the post-ribosomal supernatant of unactivated lymphocytes that could repress translation in rabbit reticulocyte lysates, but this putative inhibitor has not been characterized. The apparent regulation of eIF3j association with eIF3 and the subsequent binding of eIF3 to the 40 S ribosomal subunit may be affected by changes in the phosphorylation status of these proteins. Little is known about eIF3 subunit phosphorylation and the effects on eIF3 activity.
The in vivo binding of eIF3 to 40 S subunits has been examined in detail in the yeast Saccharomyces cerevisiae (70). Deletion analysis implicates portions of eIF3a, eIF3c, and eIF3j in stabilizing yeast eIF3 binding to 40 S subunits and improved translation (71). Interestingly, deletions that block the formation of the multifactor complex and binding of the ternary complex to the 40 S subunit nevertheless allow stable eIF3·40 S complexes to form. The results in yeast indicate that eIF3 binding to 40 S ribosomal subunits does not require stabilization by components of the multifactor complex but can occur with eIF3 alone (although roles for eIF1A and eIF1 are not ruled out). In contrast to the yeast system, human eIF3 possessing all of its subunits binds with high affinity to 40 S subunits in vitro, but eIF3 lacking only eIF3j fails to bind stably (26). Furthermore, the subcomplex, eIF3bgi, which binds 40 S subunits poorly, is stabilized on the 40 S subunit when eIF3j is added. Because intact eIF3a and eIF3c are present in the non-binding eIF3 lacking eIF3j, and are absent in the binding subcomplex, eIF3bgij, it appears that eIF3a and eIF3c do not confer essential stabilizing elements for mammalian eIF3. Instead, eIF3j may play an essential role in eIF3 binding to 40 S subunits, as well as a stabilizing role in forming the eIF3 complex.
A role for eIF3j in promoting eIF3 binding to 40 S subunits in activated T lymphocytes is suggested by the analyses reported here. In quiescent cells, eIF3j is not part of eIF3, but is unassociated with any large complexes, barely sedimenting into the sucrose gradient (Figs. 5 and 6). Upon activation of T cells, eIF3j now is found associated with eIF3 or 40 S complexes. eIF3j association with eIF3 or 40 S subunits correlates with the ability of eIF3 to bind to 40 S ribosomes and also correlates with the increase in translation rates. The in vitro experiments with purified eIF3 and eIF3j strongly support the view that eIF3j promotion of eIF3–40 S binding is defective in quiescent T lymphocytes. That the phenomenon is seen upon stimulation with I+P, but not with PMA alone, suggests that calcium regulation may be involved in "activating" eIF3j.
A role for calcium in promoting eIF3–40 S binding is supported by earlier work in Ehrlich ascites cells (64). Ehrlich ascites cells treated with a calcium chelator exhibit an inhibition of protein synthesis, which is relieved by addition of calcium. In lysates of the inhibited cells, eIF3 is no longer bound to 40 S ribosomes, but binds again when calcium is replenished. Thus, calcium appears to regulate eIF3–40 S binding in these cells. Because ionomycin (a calcium ionophore) is needed to efficiently stimulate eIF3j association with eIF3 and higher 40 S complexes in T lymphocytes, we propose that calcium-regulated signaling may affect these steps. We postulate that calcium signaling alters either eIF3j, eIF3, or 40 S subunits to enable eIF3j to associate with eIF3 or 40 S subunits and thereby promote eIF3 binding to ribosomes. Such regulation in mammalian cells has not been studied, yet it provides an intriguing area for further experimentation.
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¶ To whom correspondence should be addressed: Division of Hematology/Oncology, UC Davis Cancer Center, 4501 X St., Rm. 3016, Sacramento, CA 95817. Tel.: 916-734-3769; Fax: 916-734-6415; E-mail: smiyamot{at}ucdavis.edu.
1 The 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.
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