©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
T-cell Activation Leads to Rapid Stimulation of Translation Initiation Factor eIF2B and Inactivation of Glycogen Synthase Kinase-3 (*)

(Received for publication, December 29, 1995; and in revised form, February 20, 1996)

Gavin I. Welsh (1)(§)(¶) Suzanne Miyamoto (2) Nigel T. Price (1) Brian Safer (2) Christopher G. Proud (1)(§)

From the  (1)Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom and the (2)Molecular Hematology Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1654

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Mitogenic stimulation of T-lymphocytes causes a rapid activation of protein synthesis, which reflects in part increased expression of many translation components. Their levels, however, rise more slowly than the rate of protein synthesis, indicating an enhancement of the efficiency of their utilization. Initiation factor eIF2B catalyzes a key regulatory step in the initiation of translation, and we have therefore studied its activity following T-cell activation. eIF2B activity rises quickly, increasing as early as 5 min after cell stimulation. This initial phase is followed by an additional slow but substantial increase in eIF2B activity. The level of eIF2B subunits did not change over the initial rapid phase but did increase at later time points. Northern analysis revealed that levels of eIF2B mRNA only rose during the later phase. The rapid activation of eIF2B following mitogenic stimulation of T-cells is therefore mediated by factors other than its own concentration. The largest () subunit of eIF2B is a substrate for glycogen synthase kinase-3 (GSK-3), the activity of which rapidly decreases following T-cell activation. Since phosphorylation of eIF2B by GSK-3 appears to inhibit nucleotide exchange in vitro, this provides a potential mechanism by which eIF2B may be activated.


INTRODUCTION

Mitogenic stimulation of resting T-cells results in marked increases in rates of protein, RNA, and DNA synthesis(1) . The focus of the present study is the control of mRNA translation following T-cell activation. This stimulation is achieved by an increase in the efficiency of translation expressed relative to ribosome content and reflects mainly an increase in the rate of translation initiation (2, 3, 4, 5, 6) .

Initiation is a complex process involving at least 10 translation initiation factors (eIFs), (^1)many of which are multimeric proteins(7, 8, 9, 10) . The two major processes occurring during initiation involve the binding of the initiator tRNA (Met-tRNA) to the ribosome, which is mediated by initiation factor eIF2 and the attachment of the ribosome to the mRNA and the location of the start codon, which involves several protein factors. The activities of a number of translation factors are thought to be regulated by phosphorylation/dephosphorylation(7, 10, 11) .

In the case of eIF2, phosphorylation of serine 51 in its alpha-subunit blocks the recycling of non-phosphorylated eIF2 by the guanine nucleotide-exchange factor eIF2B, thus leading to decreased eIF2 activity. eIF2B mediates exchange of GDP bound to eIF2 for GTP, thus regenerating active (eIF2bulletGTP) complexes after each round of translation initiation(7, 9, 11) . This nucleotide-exchange step is a key control point for translation initiation. In addition to the phosphorylation of eIF2alpha, the activity of eIF2B can be regulated by several mechanisms, which include its phosphorylation (12, 13, 14, 15) and its modulation by allosteric effectors(14, 16, 17, 18, 19, 20) .

Previous work has shown that the levels of several translation factors, including eIF2alpha and the mRNA-binding factor eIF4E, are increased in T-cells following their stimulation(21, 22, 23) . Although eIF2alpha mRNA levels rise rapidly following stimulation, appreciable changes of eIF2 protein are seen only after about 10 h. However, the rate of protein synthesis rises much more rapidly than this, activation being seen as early as 15 min after stimulation(24, 25) . There is a marked shift of ribosomes into polysomes (indicative of enhanced translation initiation), which precedes significant increases in the levels of factors such as eIF2 and eIF4E, suggesting that this effect is the result of activation of pre-existing components, at least in the initial phase (see, e.g., (21) ). Evidence has been presented that this involves activation of the binding of Met-tRNA to the 40 S subunit(4) , which is mediated by eIF2 and can be regulated through changes in the activity of eIF2B.

We have therefore examined the activity of the exchange factor eIF2B at early times following T-cell stimulation and have used our anti-eIF2B antibodies to assess alterations in the cellular level of this factor. Our data show that eIF2B is rapidly and markedly activated after T-cell stimulation, without a corresponding change in its concentration. This occurs independently of changes in the level of phosphorylation of the alpha-subunit of eIF2 and indicates that an alternative mechanism operates to modulate eIF2B activity following T-cell activation. This may involve regulation of eIF2B by phosphorylation by glycogen synthase kinase-3 (GSK-3)(15) . Phosphorylation of eIF2B by GSK-3 appears to inhibit its activity in vitro, (^2)and a variety of stimuli that activate eIF2B have been shown to cause inactivation of GSK-3(15, 26, 27, 28, 29, 30, 31) . Here we show that, consistent with this idea, stimulation of T-cells causes a pronounced fall in GSK-3 activity during the first 4 h of mitogenic stimulation.


EXPERIMENTAL PROCEDURES

Materials

Chemicals and biochemicals were obtained, respectively, from BDH (Poole, Dorset, United Kingdom (UK)) and Sigma (Poole, Dorset, UK), unless otherwise indicated. Acrylamide and methylenebisacrylamide were from Pharmacia-LKB (Milton Keynes, UK). [^3H]GDP (12.4 Ci mmol) was from Amersham International (Amersham, Bucks., UK).

The anti-eIF2alpha antibody was a kind gift from the late Dr. E. Henshaw (University of Rochester, Rochester, NY).

Isolation and Induction of T-lymphocytes

Human blood lymphocytes were obtained from whole blood fractionated by continuous counterflow elutriation(32) . The preparation was further enriched for T-lymphocytes by Ficoll(TM) gradient centrifugation and passage over nylon-wool columns(22) . By fluorescence-activated cell-sorter analysis, these cells were normally found to be >90-95% T-lymphocytes. Quiescent G(o) T-cells were activated by incubation with 0.25 µM ionomycin and 10 ng ml phorbol myristate acetate in RPMI 1640 with 25 mM HEPES, 1% (w/v) glutamine, 100 µg ml penicillin, and 100 µg ml streptomycin, supplemented with 10%(v/v) fetal bovine serum. After incubation, cells were harvested and washed three times with cold phosphate-buffered saline, and the cell pellets were frozen immediately on dry ice.

Assay of eIF2B

Cell extracts were prepared and eIF2B activity was assessed as described previously(34, 35) , except that the extraction buffer contained 20 mM HEPES, pH 7.5, rather than Tris.

Electrophoresis, Isoelectric Focusing, and Immunoblotting

Prior to analysis, eIF2 and eIF2B were partially purified and concentrated from cell extracts using fast flow Sepharose-S(35) . The level of eIF2B in unstimulated and mitogenically activated T-cells was assessed by Western blot analysis (37) using a rabbit polyclonal antibody raised to a peptide whose sequence was derived from that of rabbit eIF2B. The level of eIF2alpha phosphorylation was analyzed by one-dimensional polyacrylamide gel isoelectric focusing(35, 36) . Ampholines in the pH range 4-6.5 were used. Following electrophoresis, gels were Western-blotted (37) with visualization by enhanced chemiluminescence.

Protein Kinase Assays

The activity of GSK-3 was assayed as described previously(15, 28, 38) .

Northern Blot Analysis of eIF2B mRNA Levels

Total mRNA was extracted from nylon-wool enriched T-cells activated by 0.25 µM ionomycin and 10 ng/ml phorbol myristate acetate using RNeasy(TM) (Qiagen). Samples (10 µg) were fractionated by denaturing formaldehyde-agarose gel electrophoresis, transferred to Nytran (Schleicher & Schuell), and UV cross-linked (Stratagene). Blots were hybridized with random-primed probes (Life Technologies, Inc.) washed according to the procedure of Church and Gilbert (39) and exposed to XAR film (Eastman Kodak Co.). Results were quantitated using a laser densitometer (Molecular Dynamics Personal Densitometer).


RESULTS

Activation of eIF2B Occurs with Biphasic Kinetics

The activity of eIF2B was measured following activation of primary human lymphocytes by the calcium ionophore ionomycin and the phorbol ester phorbol myristate acetate. eIF2B activity was very low in unstimulated T-cells. Following stimulation of the cells, eIF2B activity rose quickly. Increased activity was observed as early as 15 min, attaining a substantial level of activation (at least 10 fold) by 1 h. eIF2B activity continued to rise until at least 24 h after stimulation, although the rate of increase after 4 h was much less than in the first phase following cell activation. Typical data for two different types of time course are shown in Fig. 1. This increase in eIF2B activity closely parallels the increased rate of overall protein synthesis, as assessed by incorporation of [S]methionine(24, 25) .


Figure 1: Effect of mitogenic stimulation on eIF2B activity in T-cells. eIF2B activity was measured in extracts of T-cells as described under ``Experimental Procedures.'' The figure depicts two separate experiments using different preparations of T-cells in which the basal level of activity of eIF2B in the unstimulated cells differed. Panel A shows a short time course of 0-2 h following activation, while panel B shows a time course up to 24 h (panel B). Each point is the average of a duplicate assay, and the results are typical of five separate experiments in which essentially the same time courses were observed. The figure shows the activity of eIF2B as the initial rate of release of [^3H]GDP (per minute) in the standard assay. The period of activation of the T-cells is as indicated.



Similar increases in the activity of eIF2B were seen in primary human lymphocytes in response to the mitogenic lectin phytohemagglutinin (0.5 µg/ml, data not shown).

Level of eIF2B in Stimulated T-cells

The increase in eIF2B activity following T-cell activation could be due either to activation of pre-existing factor or to synthesis of new eIF2B (or a combination of both). The rapidity and magnitude of the response suggested that activation of the protein rather than an increase in its concentration was responsible. To address this point, we assessed the level of eIF2B in unstimulated and mitogenically activated T-cells by Western blot analysis using a rabbit polyclonal antibody raised to a peptide whose sequence was derived from that of rabbit eIF2B. No increase in the level eIF2B was observed during the first 4 h of mitogenic stimulation. However, a marked increase was seen between 8 and 24 h (Fig. 2). Similar kinetics have been observed previously for the levels of eIF2alpha and eIF4E in T-cells(21, 22, 23) . To examine the basis of the biphasic kinetics observed, the level of the mRNA encoding eIF2B was determined by Northern analysis (Fig. 3). During the first 4 h of stimulation, where the rate of increase in eIF2B activity is most rapid, there was little or no change in the low levels of mRNA for eIF2B. However, at later time points mRNAs levels did increase, by 6-fold at 8 h and 25-30 fold at 16-24 h.


Figure 2: Levels of eIF2B in stimulated T-cells. Extracts prepared from T-cells (stimulated, for the indicated time in hours, or unstimulated, G) were subjected to Western blotting as described under ``Experimental Procedures.'' The Western blot was developed using an anti-eIF2B antibody and visualized by enhanced chemiluminescence. The position of eIF2B is indicated. These data are typical of those obtained in four entirely separate experiments.




Figure 3: Levels of mRNA for eIF2B in T-cells. Northern blot analysis was performed on total RNA (10 µg/sample) from nylon-wool enriched T-cells activated as described under ``Experimental Procedures'' for the indicated times (hours). Blots were probed with P-labeled probe for eIF2B. Panel A, autoradiograph of Northern blot; panel B, ethidium bromide stain of gel to indicate relative loadings.



The increase in eIF2B activity during the early time period (0-4 h) therefore seems likely to be due to activation of pre-existing factor. It should be emphasized that the initial phase of the rise in protein synthesis in activated T-cells precedes the later increases in the levels of factors such as eIF2 and eIF4A(21, 23, 24, 25) , which only become significant after about 10 h or more, by which time the initial rise in eIF2B activity seen here is almost complete.

State of Phosphorylation of eIF2alpha

Since the guanine nucleotide-exchange activity of eIF2B can be modulated by the phosphorylation state of its substrate eIF2alpha(40) , we used an isoelectric focusing/immunoblotting technique to examine the level of phosphorylation of eIF2alpha following mitogenic stimulation of T-cells. The level of phosphorylation of eIF2alpha did not change up to 24 h after stimulation (Fig. 4). Furthermore, at early times there was no apparent change in the total level of eIF2, which is consistent with the data of Jedlicka and Panniers(23) . Our data indicate that the observed stimulation of eIF2B is not a consequence of a fall in the proportion of eIF2alpha in the phosphorylated form or of a drop in the absolute level of phosphorylated eIF2alpha.


Figure 4: Level of eIF2alpha phosphorylation in T-cells. The level of phosphorylation of eIF2alpha was assessed by isoelectric focusing/immunoblotting in samples from control or stimulated T-cells (time, in hours, indicated). The positions of the phospho- (alpha(P)) and dephospho- (alpha) forms of eIF2alpha are indicated.



Activity of Glycogen Synthase Kinase-3

We have reported previously that GSK-3 can phosphorylate the -subunit of eIF2B(15) . Phosphorylation by GSK-3 appears to inhibit the nucleotide-exchange activity of eIF2B in vitro.^2 We therefore studied whether GSK-3 activity changed following T-cell stimulation. Its activity fell rapidly by about 40% within 15 min of stimulation of the cells. It reached a minimum level after 4 h (approximately 50% of control) and then returned gradually to control levels by 24 h (Fig. 5, A and B).


Figure 5: Effects of stimulation on the activity of GSK-3 in T-cells. The activity of GSK-3 was assayed in T-cell extracts as described under ``Experimental Procedures'' against the standard peptide substrate based on glycogen synthase. Panels A and B show GSK-3 activity over a short (0-2 h) or long (0-25 h) time course following mitogenic stimulation of the cells. In all cases each point is the average of a duplicate assay, and the results are typical of four separate experiments in which essentially the same time courses were observed.




DISCUSSION

In this paper we present the first evidence that mitogenic stimulation of T-cells results in the activation of the guanine nucleotide-exchange factor eIF2B. This activation is rapid, preceding any change in the level of the protein (as assessed by Western blotting for its -subunit) and is likely to result from regulation of its activity by covalent modification or allosteric means (see below). We cannot, however, completely eliminate the possibility that, while the -subunit is present at constant levels, there are rises in the levels of the other subunits. This seems unlikely since there is no evidence that individual subunits exist ``free'' in the cell, but since our antibodies to other subunits of eIF2B are not sensitive enough to detect their corresponding polypeptides in T-cell extracts, even after the partial purification carried out here, we are currently unable to test this. It appears that the rise in the level of the -subunit lags behind the rise in the level of its mRNA; this is probably not surprising, since it may some time for the steady state level of the protein to increase.

Jedlicka and Panniers (23) concluded that there was no significant change in eIF2B activity within the first 8 h after T-cell activation. However, this was based on an indirect assay in which eIF2B was assayed by its ability to promote the formation of eIF2bulletGTPbulletMet-tRNA complexes rather than by the direct exchange assay used here. The former assay may not accurately reflect eIF2B activity, for example, because other factors may play a role in stabilizing ternary complexes in crude extracts. Second, these authors did not include protein phosphatase inhibitors in their extraction buffer. Under these circumstances, either eIF2B or eIF2 might undergo dephosphorylation, which would eliminate any differences in eIF2B activity due to alterations in the phosphorylation of these two proteins, each of which may be regulated by this modification (see below). It should be noted that, at later stages of T-cell activation (after about 8-10 h), the levels of both eIF2 and eIF2B increase(21, 23) . Indeed, they appear to rise roughly in parallel, such that the eIF2:eIF2B ratio (a critical parameter for determining the sensitivity of eIF2B to alterations in the level of eIF2alpha phosphorylation) is maintained roughly constant at about 5:1(23) .

Boal et al.(21) assessed the activity of eIF2 in T-cell extracts by their ability to form 43 S(N) initiation complexes (in which Met-tRNA is bound to the 40 S ribosomal subunit, mediated by eIF2) and concluded that there was no increase in the efficiency of utilization of eIF2. However, closer inspection of their data indicates that the formation of such complexes increased by about 140% between 0 and 8 h after activation, while the level of eIF2 rose by only about 12%. Thus the efficiency of utilization of eIF2 is enhanced over this time period. Their findings, for the early stage following T-cell stimulation, are at least qualitatively consistent with ours (although the degree of activation of eIF2B that we see is larger than their data would suggest).

The absence of a significant change in eIF2alpha phosphorylation argues against dephosphorylation of this protein as the mechanism by which eIF2B activity is enhanced in activated T-cells. Clearly, if the ratio of eIF2:eIF2B were very high, then only a small (and, in the extreme case, barely detectable) fall in eIF2alpha phosphorylation could lead to substantial activation of the exchange factor. However, in T-cells (at least from rat or calf) this ratio is only about 5:1, so that very substantial dephosphorylation of eIF2alpha would be required to achieve the marked (10-20-fold) activation of eIF2B observed here. It is therefore very unlikely that changes in eIF2alpha are significant in the activation of eIF2B following mitogenic stimulation of T-cells. Over a range of experiments, the level of phosphorylated eIF2alpha was between 15 and 20%, as judged by densitometric analysis of the Western blot. This is slightly lower than the value of 33% reported by Jedlicka and Panniers(23) , who, however, also found no change in the level of phosphorylation over the first 4 h following stimulation.

What other mechanisms could account for the activation of eIF2B in stimulated T-cells? There are two main possibilities, namely allosteric regulation and control by covalent modification such as phosphorylation. Although eIF2B can be allosterically activated in vitro by several compounds(14, 16, 19, 20) , it remains unclear what role, if any, they play in the control of eIF2B in vivo. In the case of the assays performed here, which involve substantial dilutions of the cell cytoplasm during the preparation of the extracts and further dilution during the assay, it is likely that the effects of such rather low affinity ligands would be lost.

A second possibility is that the activity of eIF2B in T-cells is regulated by its own phosphorylation. Our extracts are prepared in the presence of phosphatase inhibitors in order to preserve the in vivo states of phosphorylation of proteins of interest. eIF2B can be phosphorylated by at least three protein kinases (at least in vitro), namely casein kinases-1 and -2 and glycogen synthase kinase-3, GSK-3, all of which phosphorylate the largest, or -, subunit(12, 13, 14, 15) . Phosphorylation by each of these kinases may be involved in regulating eIF2B activity; the two casein kinases have each been reported to activate eIF2B(12, 13) , while phosphorylation by GSK-3 appears to inhibit eIF2B.^2 Thus, activation of either of the casein kinases or inactivation of GSK-3 could provide a mechanism for the activation of eIF2B following T-cell stimulation. It is not known whether mitogenic stimulation of T-cells alters the activity of the two casein kinases. Indeed, their regulation remains poorly documented and understood. Furthermore, in our hands phosphorylation of eIF2B by either of these two kinase has no measurable affect on its activity(14) . In contrast, it is now clear that GSK-3 is inactivated following stimulation of a variety of types of cells by agents such as insulin, growth factors, or phorbol esters (15, 26, 27, 28, 29, 30, 31) , although data were previously lacking for T-cells. Here we show that activation of T-cells results in substantial inactivation of GSK-3. The marked and prolonged inactivation of GSK-3 provides a potential mechanism by which eIF2B activity could be increased in these cells, although further confirmation of this requires analysis of the phosphorylation state of the -subunit of eIF2B in resting and stimulated T-cells. We are currently devising techniques for studying changes in the phosphorylation of this polypeptide. Our data from other cell types reveal that it is phosphorylated on multiple residues in intact cells, and this makes analysis more complex. This is especially true for cells such as primary T-lymphocytes, for which very restricted amounts are available. The upstream regulation of GSK-3 remains poorly understood. Although initial in vitro data suggested potential roles for the mitoegn-activated protein kinase and p70 S6 kinase signaling pathways(41, 42) , more recent data are not consistent with this and therefore alternative regulatory pathway(s), perhaps involving protein kinase B, appear to be involved(27, 28, 30, 43) .


FOOTNOTES

*
This work was supported by grants from the Wellcome Trust and the British Diabetic Association (to C. P.) and by the Division of Intramural Research, NHLBI, National Institutes of Health (to B. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biosciences, University of Kent at Canterbury, Canterbury CT2 7NJ, United Kingdom.

To whom correspondence should be addressed. Tel.: 44-1227-827597; Fax: 44-1227-763912.

(^1)
The abbreviations used are: eIF, eukaryotic initiation factor; GSK-3, glycogen synthase kinase-3.

(^2)
G. I. Welsh and C. G. Proud, unpublished data.


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