(Received for publication, December 29, 1995; and in revised form, February 20, 1996)
From the
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.
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), ()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
-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 (eIF2
GTP) 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 eIF2
, 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 eIF2 and the mRNA-binding factor eIF4E, are
increased in T-cells following their
stimulation(21, 22, 23) . Although eIF2
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
-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, (
)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.
The anti-eIF2
antibody was a kind gift from the late Dr. E. Henshaw (University of
Rochester, Rochester, NY).
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
[H]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).
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.
Figure 4:
Level of eIF2 phosphorylation in
T-cells. The level of phosphorylation of eIF2
was assessed by
isoelectric focusing/immunoblotting in samples from control or
stimulated T-cells (time, in hours, indicated). The positions of the
phospho- (
(P)) and dephospho- (
) forms of eIF2
are indicated.
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.
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 eIF2GTP
Met-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 eIF2
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 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
eIF2 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
eIF2
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
eIF2
would be required to achieve the marked (10-20-fold)
activation of eIF2B observed here. It is therefore very unlikely that
changes in eIF2
are significant in the activation of eIF2B
following mitogenic stimulation of T-cells. Over a range of
experiments, the level of phosphorylated eIF2
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.
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) .