(Received for publication, June 16, 1995; and in revised form, August 22, 1995)
From the
In quiescent cells high levels of protein synthesis are required
in order to re-enter the cell cycle upon stimulation. Initiation of
polypeptide synthesis is the step most often subject to regulation,
controlled in part by phosphorylation of 40 S ribosomal protein S6 and
a number of initiation factors. The kinase responsible for S6
phosphorylation is p70. We now show that the p70
pathway can be selectively blocked by the aminopurine analogue,
SQ 20006. This agent is known to raise cAMP levels, resulting in
activation of protein kinase A. We present evidence that the increase
in cAMP is not responsible for the inhibitory effect observed. We also
show that SQ 20006 can prevent the activation of p70
in a
rapid and reversible manner. The compound does not exert its inhibitory
activity on p70
but can inhibit in vitro two
protein kinase C isozymes (
and
). In a B lymphoblastoid cell
line, treatment with SQ 20006 results in inhibition of protein
synthesis at the initiation stage. In contrast, when tested directly
upon the translational machinery in the reticulocyte lysate, inhibition
is manifest at both the level of initiation and elongation. The role of
protein kinase A in the modulation of p70
and the rate of
translation is discussed.
Stimulation of cell growth and proliferation is initiated at the cell surface by specific ligand-receptor interactions. Such interactions lead to receptor dimerization, cross-phosphorylation (1) and recruitment of Src-homology 2-containing signal transducers, which dock at phosphorylated tyrosine residues(2) . This in turn causes activation of signaling molecules by a variety of mechanisms, including tyrosine phosphorylation(3) , conformational changes(4, 5) , and translocation to the plasma membrane(6, 7) . The signal is further propagated and amplified by cascades of cytosolic protein kinases(8, 9) , which ultimately activate transcription factors in order to initiate metabolic processes necessary for growth.
One obligatory step required for progression
through the cell cycle is the activation and maintenance of high rates
of protein synthesis(10) . Control of translation plays an
important role in cell proliferation (reviewed in (11) ), with
physiological regulation almost always exerted at the level of
polypeptide chain initiation(12, 13) . This phase is
regulated, in part, by the phosphorylation of initiation factors
involved in binding mRNA to the 40 S ribosomal subunit (11, 12, 13, 14, 15, 16) .
The only protein in the ribosome that has been reported to undergo
phosphorylation in vivo in response to a number of mitogens is
the 40 S ribosomal protein S6(17) . This has been mapped to an
area of the 40 S ribosomal subunit that is implicated in mRNA binding
and is thought to reside near the tRNA acceptor site(17) . S6
phosphorylation can be correlated with a selective translational
up-regulation of a family of mRNAs encoding for proteins required for
cell growth(18) . The kinase believed to modulate the level of
S6 phosphorylation is p70, which is itself activated by
phosphorylation(19, 20, 21, 23) . (
)However, the signaling pathway responsible for inducing
p70
activation remains
unidentified(24, 25) . Contrary to other
mitogen-regulated kinases, p70
activity remains high
throughout G
, and microinjection of inhibitory antibodies
at any time before S phase can block G
/S
transition(26, 27) .
The cap structure present on
the 5` end of mRNA facilitates its binding to the ribosome, a process
mediated by three initiation factors (eIF-4A, -4B, and -4F) and ATP
hydrolysis(11, 12, 13, 28, 29) .
eIF-4F is a cap binding protein complex composed of three subunits:
eIF-4E, which specifically recognizes the cap structure(13) ;
eIF-4A, an ATP-dependent, single strand RNA-binding protein with
helicase activity(13, 28) ; and eIF-4 (p220,
eIF-4G), whose function is unknown but whose integrity is required for
eIF-4F complex activity (11, 12, 13) . It is
believed that eIF-4F functions to unwind secondary structure in the
mRNA 5`-untranslated region to facilitate binding to the 40 S ribosomal
subunit(11, 12, 28, 29) . Consistent
with its proposed regulatory role, eIF-4E exists in both phosphorylated
and nonphosphorylated forms (11, 12, 13, 15) and is believed to
be the least abundant of the initiation
factors(15, 30) . In response to the appropriate
stimuli, increased levels of eIF-4E phosphorylation have been directly
correlated with increased rates of translation in a variety of cell
types (reviewed in (11) ). More recently, it has been proposed
that in adipocytes the regulated phosphorylation of an
eIF-4
-associated protein (PHAS-I, 4E-BP1) plays a role in
modulating the availability of eIF-4E to enter the initiation
pathway(31, 32) . However, the role of phosphorylation
of eIF-4E in this interaction is at present poorly defined, and
moreover, it is not known whether such interactions occur in other cell
types.
These observations have prompted us to investigate the role
of p70 in G
progression and in the
phosphorylation of eIF-4E and its association with PHAS-I. Here we
report on the properties of a p70
-specific inhibitor, SQ
20006; we show that SQ 20006 prevents the mitogen-induced activation of
p70
and causes inhibition of protein synthesis initiation
in Swiss 3T3 cells and Raji cells. SQ 20006 blocks entry of cells into
S phase but does not affect the steady state phosphorylation of eIF-4E
or its association with PHAS-I (4E-BP1).
To analyze p70 and ERK2, equal amounts of total
protein (5 µg) were resolved on SDS-PAGE as
described(20, 40) , and proteins were transferred to
polyvinylidene difluoride (Millipore). The resulting membrane was
decorated with either anti-ERK2 polyclonal antibody (40) or
with the p70
M1 antibody (20) and revealed using
the ECL system (Amersham Corp.). In order to assay protein kinase
activity, equal amounts of total protein (50 µg) were
immunoprecipitated with the p70
antibody M5 (20) or with an anti-ERK2 polyclonal antibody (40) and
immobilized on protein A-Sepharose beads. Pellets were washed with 3
1 ml of ice cold Buffer B followed by 1 ml of ice-cold Buffer C
(50 mM Tris-HCl, pH 7.5, 10 mM MgCl
, 1
mM dithiothreitol, 0.1% Triton X-100). Finally, pellets were
dried and resuspended in 20 µl of ice cold Buffer C. Kinase
activity was assayed in Buffer C in a final volume of 25 µl
containing 50 µM [
-
P]ATP
(Amersham Corp.) (specific activity, 3.2 µCi/nmol), and 50
µM peptide substrate. The synthetic peptides employed were
S6
for assaying p70
activity and
p70
for assaying ERK2
activity(19) . Reactions were terminated by the addition of
EDTA/adenosine to a final concentration of 20 mM and 1.5
mM, respectively. Incorporation of
[
P]phosphate onto the peptide substrate was
determined by spotting 20-µl aliquots on P81 paper (Whatman) as
described(41) . In the case of cyclin-dependent kinase 2,
immunoprecipitation was carried out on 300 µg of total protein in
Buffer D (25 mM Tris-HCl, pH 7.5, 60 mM
-glycerophosphate, 15 mM MgCl
, 15 mM EGTA, 0.1 mM NaF, 15 mMp-nitrophenyl
phosphate, 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride, 0.1% Nonidet P-40), and immobilized on
protein A-Sepharose beads. Pellets were washed with 3
1 ml of
ice-cold Buffer D and 1 ml of ice-cold Buffer E (50 mM Tris-HCl, pH 7.5, 10 mM MgCl
, 1 mM
dithiothreitol). Pellets were processed for kinase activity as
described for ERK2 with the exception that Buffer E was employed and
[
-
P]ATP was at a final concentration of 5
µM (specific activity, 50 µCi/nmol).
Figure 1:
SQ 20006 inhibits
the activation of p70 but not ERK2 in Swiss 3T3 cells.
Quiescent Swiss 3T3 fibroblasts were stimulated with 10% fetal bovine
serum in the presence (
) or absence (
) of 1 mM SQ 20006. Extracts were prepared at 0, 0.5, 1, 2, 4, 6, 8, and 10
h following stimulation, and either p70
(A) or
ERK2 (B) was immunoprecipitated and assayed as described under
``Experimental Procedures.'' Panels C and D, total cell extract corresponding to the time points in panels A and B was subjected to SDS-PAGE and
immunoblot analysis, employing a p70
or an ERK2
polyclonal antibody, respectively, as described above. Absence or
presence of SQ 20006 is denoted by - and +,
respectively.
Figure 2:
Rate of p70 inhibition and
recovery from inhibition by SQ 20006. Panel A, quiescent Swiss
3T3 cells were stimulated with serum for 1 h prior to the addition of 1
mM SQ 20006. Cell extracts were prepared at 0, 5, 10, 20, 30,
and 60 min (lanes 2-7) after addition of the drug, and 5
µg of total protein for each time point was analyzed on SDS-PAGE.
Blots were probed with antibody M1 (20) and revealed using the
ECL system. p70
detected in quiescent cells is shown in lane 1. Panel B, quiescent Swiss 3T3 cells were
stimulated with serum for 1 h in the presence of 1 mM SQ 20006 (lane 2). Cells were then washed to remove the drug and
resuspended in fresh medium. At 1, 2, and 3 h (lanes
3-5), cell extracts were prepared and processed as in panel A. p70
from either quiescent cells or
cells stimulated for 1 h with serum is shown in lanes 1 and 6, respectively.
Figure 3: SQ 20006 treatment prevents entry into S phase in Swiss 3T3 cells after serum starvation/stimulation. Quiescent Swiss 3T3 fibroblasts were stimulated with serum, and progression into the cell cycle was scored by flow cytometric measurement of the DNA content at 8, 12, 16, 20, 24, and 32 h (panels A-F); cells shown in panel G were incubated for 20 h in the presence of 5 µg/ml aphidicolin, and those in panel I were incubated for the same time in the presence of 1 mM SQ 20006. Asynchronous cultures of Swiss 3T3 cells treated for 24 h with 1 mM SQ 20006 are shown in panel H.
In order to investigate in more
detail the inhibition of S phase entry following SQ 20006 addition, we
first considered the response of cells to increasing doses of the drug. Fig. 4shows that the concentration of SQ 20006 required to
inhibit S phase entry by 50% (IC) is 0.2 mM, with
full block at concentrations of 0.5 mM and above (Table 2). We have also compared the effect of SQ 20006 addition
with that of a potent inhibitor of the p70
activation
pathway, the immunosuppressant, rapamycin. As shown in Table 2,
rapamycin decreases the extent of S phase transition to 48% in Swiss
3T3 cells, whereas SQ 20006 fully blocks DNA synthesis. One possible
explanation for the inhibitory effect of SQ 20006 could be that it is
due to inhibition of phosphodiesterase and a subsequent rise in the
levels of cAMP. To test this, cells were incubated in the presence of
8-Br-cAMP. As shown in Table 2, 1 mM 8-Br-cAMP did not
block entry of cells into S phase whether added at the start of the
incubation or at any time during the G
period (data not
shown). To complement these studies, aliquots of cells treated as above
for 1 h were also analyzed for the activation state of
p70
. While the addition of SQ 20006 (Fig. 2) or
rapamycin (Fig. 5) to serum-stimulated cells prevented the
activation of p70
, neither 8-Br-cAMP nor the
phosphodiesterase inhibitor, IBMX was effective at altering the
activity of p70
(Fig. 5).
Figure 4:
Dose-response effect on S phase entry by
SQ 20006. Increasing amounts of SQ 20006 were added to quiescent Swiss
3T3 fibroblasts at the time of stimulation with serum. To estimate DNA
synthesis, at 14 h (mid S phase) cells were pulse-labeled with 1
µCi of [H) thymidine for 2 h and harvested as
described under ``Experimental Procedures.'' The data are
representative of those obtained in three separate
experiments.
Figure 5:
Rapamycin, but not 8-Br-cAMP or IBMX
inhibit the activation of p70. Quiescent cells were
stimulated with phosphate-buffered saline (lane 1) or serum (lanes 2-5) for 1 h, in the absence (lanes 1 and 2) or presence of 1 mM IBMX (lane
3), 20 ng/ml rapamycin (lane 4), or 1 mM
8-Br-cAMP (lane 5). Cell extracts were prepared and
p70
activation was then monitored by SDS-PAGE, as
described.
Next, we addressed
the question of whether p70 and protein synthesis
inhibition by SQ 20006 after the restriction point (47) are
critical to S phase transition. For these studies, quiescent cells were
induced to grow by the addition of serum, SQ 20006 was added at various
times, and DNA synthesis was monitored at 14 h following stimulation. Fig. 6A shows that induction of DNA synthesis was very
sensitive to the presence of SQ 20006 during the first 10 h following
serum stimulation, with sensitivity decreased by 12 h. The addition of
SQ 20006 to cells during S phase transition (i.e. 12-18 h)
did not significantly affect the extent of
[
H]thymidine incorporation into DNA (data not
shown). Accordingly, withdrawal of the drug early in G
allowed cells to regularly proceed to S phase, whereas later
removal caused a delay in the transition to S (Fig. 6B and Table 3).
Figure 6:
The effect of time course of addition and
withdrawal of SQ 20006 on S phase entry. Panel A, 0.5 mM SQ 20006 was added to quiescent cells at 0, 2, 4, 6, 8, 10, and 12
h (lanes 2 to 8) following stimulation with serum.
[H]thymidine was added at the beginning of the
incubation, and cells were harvested at 14 h. Values are expressed as
percentage of [
H]thymidine incorporation relative
to untreated cells (lane 1). Panel B, 0.5 mM SQ 20006 was added to quiescent cells at the time of stimulation
with serum. The drug was then removed by washing aliquots of cells and
replacing the medium at 0, 2, 4, 6, 8, 10, and 12 h (lanes
2-8). Cells were labeled and harvested as described in panel
A. Lane 1, untreated cells.
Since SQ 20006 yields an apparent
G/S block, we decided to test whether the drug might also
function in a manner similar to known inhibitors of S phase entry. This
is the case for hydroxyurea, an inhibitor of the DNA precursor pool
synthesis. Cells were restimulated with serum for 15 h, in the absence
or presence of either SQ 20006 or hydroxyurea, the drugs were removed
by washing the cells, and DNA synthesis was measured at 2-h intervals
during the following 20 h by pulse labeling with
[
H]thymidine, as described. The data presented in Table 3show that release of the cells from the hydroxyurea block
was rapid, with cells progressing into S phase within 4 h. On the
contrary, cells released from SQ 20006 block required 15 h to reach
mid-S phase, independent of whether SQ 20006 was added at the start of
the incubation (Table 3) or during G
(data not
shown). Taken together, the data above suggest that SQ 20006 is not
directly involved in the inhibition of DNA replication but rather it
blocks early in G
and in a reversible manner.
Figure 7:
SQ 20006 inhibits protein synthesis
initiation in Raji cells, but does not affect the phosphorylation
status or association of eIF-4E with PHAS-I. Panel A, cells in
the mid-log phase of growth were incubated for 20 h in the absence or
presence of SQ 20006 (left panel) or rapamycin (right
panel) at the final concentrations indicated in the figure. Protein synthesis () was estimated by the
addition of 1 µCi/ml [
S]methionine for 4 h
prior to harvesting as described. DNA synthesis (
) was estimated
by [
H]thymidine incorporation in the same manner. Panel B, Raji cells (1
10
) were incubated
in the absence or presence of 0.56 mM SQ 20006 for 24 h, and
samples were prepared and analyzed for polysome profiles as described
under ``Experimental Procedures.'' Arrows indicate
the sedimentation of the 80 S ribosome. Panel C, Raji cell
extracts were prepared as in panel B, and eIF-4E was isolated
as described under ``Experimental Procedures.'' Proteins
recovered from the affinity resin were subjected to SDS-PAGE, resolved
proteins were transferred to polyvinylidene difluoride, and eIF-4E and
PHAS-I were identified using specific antiserum. Panel D,
samples prepared as in panel C to enrich for eIF-4E were
subjected to one-dimensional vertical slab isoelectric focussing and
immunoblot analysis with antiserum specific for eIF-4E, as described.
The migration of the phosphorylated form of eIF-4E is
indicated.
Considering the differences in the magnitude of inhibition observed
between SQ 20006 and rapamycin, we asked whether SQ 20006 might act on
translational targets other than p70. One potential site
of regulation is initiation factor 4E (eIF-4E), which is a limiting
factor in the process of initiation and has been demonstrated to
undergo phosphorylation in response to numerous growth factors and
hormones in a variety of cells(11, 12, 13) .
Recently it has been suggested that, in addition to phosphorylation,
interaction of eIF-4E with other proteins, such as PHAS-I
(4E-BP1(31, 32, 49) ) may play a role in
translational control. Therefore, we have looked at the association
between eIF-4E and PHAS-I following treatment of cells with SQ 20006,
by isolation of the former on m
GTP-Sepharose, as described
under ``Experimental Procedures.'' As shown in Fig. 7C, prolonged treatment of Raji cells with SQ
20006 did not affect the interaction of eIF-4E with its inhibitory
partner PHAS-I. We have also examined the phosphorylation status of
eIF-4E by vertical slab isoelectric focusing and immunoblotting. Fig. 7D shows that SQ 20006 has no effect on the steady
state phosphorylation of eIF-4E. In order to test whether SQ 20006 has
a direct inhibitory effect on the translational machinery, we have also
employed the reticulocyte lysate translation system. This in vitro system is unique, as it maintains high levels of protein
synthesis, which is largely independent of any requirement for S6
phosphorylation; indeed, p70
activity in the reticulocyte
lysate is low relative to that found in mitogen-stimulated cells. (
)As shown in Fig. 8A, time course and
dose-response experiments indicate that SQ 20006 displayed a
significant inhibition of translation at concentrations comparable with
those used above. Analysis of polysomes by sucrose density gradient
centrifugation (Fig. 8B) shows that SQ 20006 induces a weak
and incomplete disaggregation of ribosomes from polysomes and a rise in
the content of free 80 S ribosome couples. This was also seen in the
presence of the elongation inhibitor, emetine, suggesting that part of
the inhibitory effect of SQ 20006 is due to activation of low levels of
nuclease. However, this level of nuclease is insufficient to account
for the large inhibition of translation shown in panel A (data
not shown). An alternative conclusion is that SQ 20006 induces a weak
inhibition at the level of initiation, with a dominant effect at the
level of elongation, the latter possibly through activation of eEF-2
kinase via protein kinase A(50) . As with the Raji cells, SQ
20006 had little or no effect on the association between eIF-4E and
PHAS-I; neither did it affect the steady state phosphorylation of
eIF-4E (Fig. 8, C and D). The inhibition of
translation in the reticulocyte lysate appears to be an in vitro effect; treatment of intact reticulocytes (37) with 1
mM SQ 20006 for 90 min did not affect the rate of translation
in derived lysates, polysome disaggregation, the association between
eIF-4E and PHAS-I, or the steady state phosphorylation of eIF-4E (data
not shown). This possibly reflects the lack of requirement of this
translation system for S6 phosphorylation and activation of
p70
.
Figure 8:
SQ 20006 inhibits translation in the
reticulocyte lysate. Panel A, left side, reticulocyte
lysate translation assays as described under ``Experimental
Procedures'' were carried out in the absence () or presence
of 0.5 mM (
) or 1 mM (
) SQ 20006, and
[
S]methionine incorporation into protein was
determined as above. Panel A, right side. Translation
assays were carried out as described for 45 min in the absence or
presence of the final concentrations of SQ 20006 as indicated.
Incorporation of labeled amino acid into protein was determined and is
expressed as the percentage of incorporation in the absence of SQ
20006. Error bars are S.D. (n = 3). Panel
B, reticulocyte lysate was incubated in the absence (left) or presence of 1 mM SQ 20006 (middle)
or presence of 1 mM SQ 20006 and emetine (right) for
30 min, and samples were prepared and analyzed for polysome profiles as
described. Arrows indicate the sedimentation of the 80 S
ribosome. Panel C, reticulocyte lysate was incubated in the
absence or presence of SQ 20006 for 30 min, prior to analysis of the
association of eIF-4E and PHAS-I, as described in the legend to Fig. 7. Panel D, reticulocyte lysate, incubated as in panel C, was subjected to VSIEF analysis, as described in the
legend to Fig. 7. The migration of the phosphorylated form of
eIF-4E is indicated.
In many cell systems examined, stimulation of quiescent cells
to re-enter the cell cycle with serum or growth factors causes an
immediate drop in the intracellular concentration of cAMP(51) .
Concomitant with this event is the activation of kinase cascades, which
mediate the mitogenic effect of growth
factors(11, 17) , leading to evidence of a negative
correlation between higher levels of cAMP and mitogenesis. This has
been shown in studies that have addressed the role of the
cAMP-dependent protein kinase (protein kinase A) during cell cycle
progression in yeast(52) , during meiotic maturation of Xenopus oocytes (53) and directly on intracellular
signaling pathways(54) . A number of hypotheses have been
proposed to explain the mechanism by which raised levels of cAMP yield
a G block; these include inhibition of activation of the raf kinase (54) and increased levels of
p27
, which in turn inhibits cyclin-dependent kinase 4
activity(55) .
Recent data obtained using a lymphoid cell
line has indicated another target of protein kinase A to be the
inactivation of p70(56) . On the other hand,
previous data on S6 phosphorylation in Swiss 3T3 fibroblasts have shown
that changes in total intracellular levels of cAMP were not involved in
the inhibition of either S6 phosphorylation or protein synthesis
initiation(43) . The data presented in Fig. 1show that
in Swiss 3T3 cells, SQ 20006 is a potent inhibitor of the pathway
leading to p70
activation, although not a direct
inhibitor of the activated kinase itself in vitro (Table 1). This effect is fairly specific, as SQ 20006
selectively blocks the p70
pathway without affecting the
activation of the ERK2 kinase cascade (Fig. 1, C and D) or directly affecting the activity of ERK2 in vitro (Table 1). SQ 20006 is a potent inhibitor of
phosphodiesterase activity(42) , leading to enhanced levels of
cAMP and activation of protein kinase A. Considering that it also
inhibits p70
activation, one might be tempted to draw a
parallel between the two phenomena(56) . However, this may not
be the case in Swiss 3T3 cells, as addition of the membrane-permeable
analogue 8-Br-cAMP to cells at the time of restimulation (Fig. 5) or at any following time (data not shown) did not
affect p70
activity. Accordingly, the addition of the
phosphodiesterase inhibitor, IBMX, was also ineffective in this respect (Fig. 5). Therefore, it appears that the elevation of cellular
levels of cAMP alone is insufficient to explain the prevention of the
serum-induced activation of p70
. In Swiss 3T3 cells,
p70
is activated in a biphasic manner in response to
growth factor stimulation, with the second phase under the control of
protein kinase C(44) . When SQ 20006 was tested in vitro on several kinases, it appeared to be particularly effective in
inhibiting protein kinase C
and protein kinase C
(Table 1). These data suggest that protein kinase C
and/or
protein kinase C
might function upstream of p70
, or
else a still, as yet unknown, p70
kinase(s) might be
target for inhibition by SQ 20006.
Contrary to other mitogen-induced
protein kinases (such as ERK2; Fig. 1), p70 activity remains high throughout G
, and
microinjection of inhibitory antibodies at any time in G
can block the G
/S
transition(26, 27) . Considering the low turnover rate
of SQ 20006 and that it was not toxic to cells, we set out to examine
whether prolonged inhibition of p70
was involved in
blocking transition to S phase. As shown by FACScan analysis, SQ 20006
addition to cells yields an apparent G
/S block, similar to
that seen with aphidicolin ( Fig. 3and Table 2), which
could be reversed by removing the drug (Table 3). We conclude
that the inhibition set by SQ 20006 cannot be bypassed and does not
allow cells to progress to S phase. In similar experiments, the
immunosuppressant rapamycin is known to cause only a delay in the
transition to S phase despite full inhibition of p70
activity ( Table 2and Refs. 20, 22, and 57). Furthermore,
we observed that release from SQ 20006 did not allow a rapid initiation
of DNA replication. Cells appeared to require at least 10 h to enter S
phase (Table 3), indicating that SQ 20006 is an early G
blocker. Treatment of cells with SQ 20006 at different times
following restimulation with serum (Fig. 6A) showed
that the compound could set an effective block only before the
restriction point(47) . In agreement with published data, this
implies that p70
function is necessary throughout the
G
phase of the cell cycle(26) . Accordingly,
withdrawal of the drug from the medium within the first 2 h of exposure
allowed cells to proceed to S phase, whereas removal at times after 4 h
did not prevent the inhibitory effect of SQ 20006 on the transition to
S phase (Fig. 6B and Table 3). These data suggest
that as a result of, or as a consequence of, the inactivation of
p70
, cells are returned to an early point in
G
, possibly to enable the cells to resynthesize labile
proteins necessary for G
progression. Indeed,
immunoprecipitation of cyclin-dependent kinase 2 with anti-cyclin E
antiserum indicated a dramatic drop in cyclin-dependent kinase 2
activity 10 h following serum stimulation in the presence of SQ 20006
(data not shown) without affecting the activity of this kinase in
vitro (Table 1).
Early studies with SQ 20006 in Swiss 3T3
cells showed that the addition of the drug decreased the mobilization
of 80 S ribosomes into polysomes(43) . This is indicative of a
lesion at the level of polypeptide chain initiation. We now show that
SQ 20006 can inhibit translation initiation in the B lymphoblastoid
cell line, Raji, when maintained in the mid-log phase of growth (Fig. 7A, left panel). The effect of SQ 20006 on
translation was more pronounced than that seen with rapamycin (Fig. 7A, right panel). Because both SQ 20006 and
rapamycin inhibit the activation of p70, we have studied
other aspects of protein synthesis initiation in an attempt to explain
the different effects of these compounds on protein synthesis. In
mammalian cells, there is evidence for the regulation of translation by
phosphorylation of initiation factors and their associated proteins
(PHAS-I, 4E-BP1) involved in binding mRNA to the 40 S ribosomal subunit
(11-13, 16, 29, 31, 32, 49). Although S6 phosphorylation may play
a role in the selective binding of mRNA species to
ribosomes(17, 18, 48) , the activity of
initiation factors, such as the eIF-4F complex, may also influence the
selection of mRNA from the cellular pool for
translation(11, 12, 13) . It is believed that
the eIF-4F complex functions to unwind secondary structure in the mRNA
5`-untranslated region to facilitate binding of the 40 S ribosome. As
with S6, in response to the appropriate stimuli, increased levels of
eIF-4E phosphorylation have been directly correlated with increased
rates of translation in a variety of cell
types(11, 12, 13) . The data presented in Fig. 7and Fig. 8show that SQ 20006 affected neither the
association between eIF-4E and PHAS-I nor the phosphorylation status of
eIF-4E in Raji cells or reticulocyte lysate, respectively. Together
these data confirm the finding that eIF-4E lies on a signaling pathway
distinct from that of p70
(39) and suggest that
protein kinase C
and/or protein kinase C
is not involved in
the serum-stimulated phosphorylation of eIF-4E in vivo. At
this time, the exact function of the coordinate phosphorylation of S6
and eIF-4E in recruiting mRNA to the ribosome is not understood,
although each appears to be mediated by separate signaling pathways.