(Received for publication, July 13, 1995; and in revised form, October 23, 1995)
From the
We show here using synchronized Swiss mouse 3T3 fibroblasts that
p70 S6 kinase (p70) and mitogen-activated
protein kinases
(p42
/p44
) are not only
activated at the G
/G
boundary, but also in
cells progressing from M into G
. p70
activity increases 20-fold in G
cells released
from G
. Throughout G
, S, and G
it
decreases constantly, so that during M phase low kinase activity is
measured. The kinase is reactivated 10-fold when cells released from a
nocodazole-induced metaphase block enter G
of the next cell
cycle. p42
/p44
in
G
cells are activated transiently early in G
and are reactivated late in mitosis after nocodazole release.
p70
activity is dependent on permanent signaling
from growth factors at all stages of the cell cycle. Immunofluorescence
studies showed that p70
and its isoform
p85
become concentrated in localized spots in
the nucleus at certain stages in the cell cycle. Cell cycle-dependent
changes in p70
activity are associated with
alterations in the phosphorylation state of the protein. However,
examination of the regulation of a p70
mutant in
which the four carboxyl-terminal phosphorylation sites are changed to
acidic amino acids suggests that a mechanism independent of these
phosphorylation sites controls the activity of the enzyme during the
cell cycle.
Signaling pathways that operate through tightly controlled
protein phosphorylation cascades transduce extracellular signals to
various intracellular targets. Some of these targets regulate the
transcriptional and translational machinery to ensure proper cell cycle
progression, cell growth, or differentiation. Phosphorylation of the S6
protein of 40 S ribosomal subunits is a highly conserved response of
animal cells to treatment with growth factors, steroid hormones,
phorbol esters, and oncogenes(1) . Inhibition of S6
phosphorylation by exposure of cells to the immunosuppressant rapamycin
selectively suppresses the translation of certain mRNAs that contain a
polypyrimidine tract at the 5` end(2) . These mRNAs encode
ribosomal proteins and protein synthesis elongation factors, whose
production is required for efficient transit through the G phase of the cell cycle.
Two families of mitogen-stimulated S6
kinases have been identified: the rsk-encoded M 85,000-92,000 S6 kinases ((3) , referred to as
p90
) (
)and the M
70,000 and 85,000 S6 kinases (Refs. 4 and 5, referred to as
p70
and p85
). A variety
of evidence indicates that p90
and
p70
/p85
lie on different
signaling pathways (6, 7, 8, 9, 10) . Unlike
p70
(8) , p90
is
phosphorylated and activated by the erk-encoded M
42,000 and 44,000 mitogen-activated protein
(MAP) kinases ((6) , referred to as p42
and p44
) in response to signals
transmitted through p21
,
p74
, and
p47
(11) . Once activated,
p90
and
p42
/p44
can be
translocated to the nucleus(12, 13) , where they are
thought to phosphorylate nuclear transcription factors, thus promoting
the transcription of genes required for the growth response.
p70 is the physiological S6 kinase activity
in mammalian cells(14) . p85
is a minor
species that is identical to p70
except for the
presence of a 23-amino acid extension at the amino terminus that
carries features of a nuclear targeting
sequence(15, 16) . Indeed, p85
is localized in the nucleus (17, 18) ,
where it might phosphorylate a nuclear pool of S6 protein (19) or the cAMP-response element modulator (CREM), a
transcription factor which was recently identified as a substrate of
p70
(20) . Activation of p70
in response to mitogens is associated with phosphorylation
of three serines and one threonine located at the carboxyl terminus of
the kinase(21) . p70
also contains
additional phosphate groups that become dephosphorylated upon rapamycin
treatment, leading to inactivation of p70
(22) . Each set of phosphorylation sites might be
modified by distinct kinases; however, direct activators of
p70
/p85
are so far
unknown. Recent experiments based on the use of rapamycin (23, 24) , specific phosphatidylinositol 3-kinase
inhibitors such as wortmannin (25, 26) and
platelet-derived growth factor receptor mutants (27) have
suggested that phosphatidylinositol 3-kinase and the structurally
related enzyme RAFT/FRAP are involved in upstream signaling to
p70
/p85
.
Two lines of
evidence have suggested that the function of
p70/p85
during G
is important for cell cycle progression. First, inhibition of
p70
/p85
by treatment of
cells with rapamycin leads to cell cycle arrest in G
or a
delay of entry into S phase, depending on the cell
type(9, 14) . Second, microinjection of rat embryo
fibroblasts with antibodies that inhibit
p70
/p85
abolishes the
serum-induced entry into S phase(17, 28) . Activation
of p42
/p44
is also
thought to be essential for triggering the proliferative response in
fibroblasts(29) . To gain further insight into how
p70
/p85
and
p42
/p44
activity is
regulated and what role the enzymes might have in cell growth and cell
cycle control, we have examined the behavior of these enzymes during
the cell cycle. We show here that p70
and
p42
/p44
activities are
regulated in a cell cycle-dependent manner. Furthermore, we present
evidence that the cell cycle regulation of
p70
/p85
activity might
involve compartmentation and a regulatory mechanism that is independent
of the four carboxyl-terminal phosphorylation sites. Finally, our
observations of the behavior of p70
and
p42
/p44
during the cell
cycle suggest that there is cross-talk between these signaling
molecules and the cell cycle machinery.
To arrest cells in
metaphase, cells were first presynchronized in G by serum
starvation and then stimulated with EGF, insulin, and FCS as described
above. Nocodazole (0.4 µg/ml, Sigma) was added 20 h after release
from G
, before cells entered M phase. Mitotic cells were
collected 4 h later by gentle pipetting and were reseeded into DMEM
plus 10% FCS at 0.5
10
cells per 10-cm plate. These
conditions have been shown to result in an efficient and reversible M
phase block that prevents cells from entering a polyploid
state(30) . Mitotic cells were isolated without nocodazole by
multiple rounds of mechanical shake-off alone (31) from
subconfluent plates of cells which were synchronized in G
and stimulated for 22 h with mitogens as described above. Cells
collected from early rounds were stored in DMEM plus 10% FCS at 4
°C during the collection process.
S6 kinase activity was
measured using 40 S ribosomal subunits as substrate as described
earlier(26) . The reactions were stopped and the samples were
subjected to polyacrylamide gel electrophoresis and scintillation
counting as described previously(33) . A unit is defined as the
amount of kinase incorporating 1 pmol of P into S6 per min.
Polyclonal antibodies to a His-tagged fragment of rat p70 (amino acids 258-469; (4) ) were produced and
affinity purified as described previously(34) . These purified
antibodies show no cross-reaction with p90
on Western
blots or in immunoprecipitation kinase assays (data not shown). For
immunocomplex S6 kinase assays, Triton X-100 (to 1%),
phenylmethylsulfonyl fluoride (to 0.1 mM), and purified S6
kinase antibody (1 µl) were added to 50 µl of cell extract
supernatant and kept on ice for 3 h. Then 25 µl of 50% (v/v)
protein A-agarose (preincubated with 1% bovine serum albumin in
extraction buffer plus 1% Triton X-100) was added and incubated for 1 h
at 4 °C. The beads were washed twice with extraction buffer plus 1%
Triton X-100 and twice with S6 kinase assay buffer without
dithiothreitol. The kinase assays were performed essentially as
described above. Immunocomplex S6 kinase assays with hemagglutinin (HA)
antibody 12CA5 (35) were performed essentially as described
above except that cell extract supernatants containing 800 µg of
protein were used per sample and more extensive washes of the beads
were done.
For MAP kinase immunocomplex assays, immunoprecipitations
with antibody 122 against p42 were performed essentially
as described above, except that the last two washes were done with MAP
kinase assay buffer (30 mM Tris, pH 8, 20 mM MgCl
, 2 mM MnCl
, 0.1% Triton
X-100, and 0.1 mM dithiothreitol). MAP kinase assays were
initiated by adding 15 µl of MAP kinase assay buffer containing 10
µM ATP, 2 µM of the peptide inhibitor of
cAMP-dependent protein kinase (Sigma), 10 µg of myelin basic
protein (Sigma), and 0.33 µl [
-
P]ATP.
After 30 min at 37 °C the reactions were stopped and the samples
were subjected to electrophoresis on SDS-20% polyacrylamide gels,
autoradiography and scintillation counting.
Figure 1:
Behavior of p70 and p42
/p44
in synchronized cells released from G
.
Subconfluent fibroblasts were serum starved and released into the cell
cycle by adding mitogens at t = 0 h (see
``Materials and Methods''). A, synchrony of the
cells was followed by analyzing their DNA content by flow cytometry. B, at the indicated times after release, extract supernatants
were prepared from a parallel set of cells and immunocomplex kinase
assays were performed to measure p70
(
)
and p42
(
) activity. C,
proteins in cell extract supernatants were separated by polyacrylamide
gel electrophoresis and transferred onto nitrocellulose.
p70
(upper panel) and MAP kinases (lower panel) were detected with polyclonal antibodies (see
``Materials and Methods'').
To measure p70 activity
in extracts of synchronized cells, immunocomplex kinase assays were
performed using 40 S ribosomal subunits as a substrate. In these
experiments an antibody that recognizes both p70
and
p85
was used. However, since p85
is much
less abundant than p70
in these cells(17) , most
of the activity measured in the immunocomplexes is contributed by
p70
. p70
activity measured 20 min after
mitogen stimulation was more than 20 times higher than the activity
measured in serum-starved cells (Fig. 1B, closed
circles). The kinase lost 40% of its activity by the end of
G
(Fig. 1B, 16 h), consistent with
published data(28) . During S and G
the activity
continued to decrease, so that during M phase a relatively low level of
activity was measured (Fig. 1B). It was shown earlier
that p70
is activated at the first G
/M
boundary during meiotic maturation of Xenopus laevis oocytes(39) . However, we saw no increase in p70
activity in fibroblasts cycling from G
into M (Fig. 1B). As the nuclear transcription factor
CREM
has been shown to be phosphorylated by
p70
(20) , we also performed immunocomplex kinase
assays with recombinant CREM
protein as a substrate. The pattern
of p70
activity toward CREM
during the cell cycle
was virtually identical to that seen toward S6 (data not shown).
All
evidence obtained so far indicates that p70 is activated
by phosphorylation(21, 40) . To determine if the
gradual decrease in p70
activity observed in Fig. 1B could be due to dephosphorylation, the
phosphorylation state of p70
was assessed by its
appearance as multiple bands on Western blots. The active,
phosphorylated form of p70
migrates more slowly in
SDS-polyacrylamide gels than the dephosphorylated, inactive species (40) . After 20 min of stimulation the most highly
phosphorylated form of p70
was detected (Fig. 1C, upper panel), which correlated to
the highest S6 kinase activity (Fig. 1B, closed
circles). The hyperphosphorylated form was seen throughout
G
, and by the end of G
partially
dephosphorylated forms of the kinase appeared (Fig. 1C, upper panel). As p70
activity continued to
decrease, the hypophosphorylated forms of the kinase became
predominant. The Western analysis also showed that the amount of
p70
protein remained constant during the cell cycle (Fig. 1C). Together, these data demonstrate that
p70
activity is cell cycle regulated and that the changes
in activity appear to be mediated by phosphorylation-dephosphorylation.
In parallel to p70, we also examined the cell cycle
regulation of MAP kinases. Immunocomplex kinase assays showed that
p42
was strongly but only transiently activated early in
G
/G
(Fig. 1B, open
squares). During S and G
, p42
activity
was at basal levels and during G
/M the kinase activity
increased to a small extent. MAP kinases were also visualized on
Western blots that were probed with an antibody that recognizes both
p42
and p44
(Fig. 1C, lower panel). The mitogen-dependent activation of
p42
/p44
was clearly seen as a shift of
the proteins to the phosphorylated, more slowly migrating forms after
20 min of stimulation (Fig. 1C, lower panel).
Phosphorylated species of p42
/p44
were
also seen at 4 h but thereafter the dephosphorylated forms were
predominant. Because p42
gives a relatively faint signal
on Western blots, no up-shifted band that would represent active
p42
was seen in G
/M extracts (Fig. 1C, lower panel). However, a minor upper
band representing active p44
was visible at later stages
in the cell cycle (Fig. 1C, lower panel).
Figure 2:
p70 and
p42
/p44
in cells
progressing from M phase into G
. Mitotic cells were
collected after nocodazole treatment as described under
``Materials and Methods'' and reseeded into drug-free medium
at t = 0 h. A, entry into G
was
followed by analyzing cellular DNA content by flow cytometry. B, at the indicated times, cell extract supernatants were made
and p70
(
) and p42
(
) activity was measured in immunocomplex kinase assays. C, the amount of expressed protein and the phosphorylation
state of p70
(upper panel) and MAP
kinases (lower panel) were determined by Western
analysis.
p42 also became activated in G
cells
after release from nocodazole but the activity returned very rapidly to
near-basal levels (Fig. 2B, open squares).
Western analysis showed that p42
and p44
were phosphorylated 1.5 h after release from nocodazole and
extensively dephosphorylated 3 h after release (Fig. 2C, lower panel). These results show
that p70
and p42
/p44
are
not only activated at the G
/G
boundary, but
also in fibroblasts cycling from M into G
after release
from a nocodazole block.
One could argue that the low p70 activity measured in metaphase-arrested cells might be an
artifact of nocodazole treatment. To exclude this possibility, we
compared the amount of p70
activity in mitotic cells that
were collected by shake-off with or without nocodazole treatment and
found that there was no significant difference (Fig. 3A). In addition, there was no difference in
kinase activity in the drug-treated or non-treated cells that were left
on the plates after mitotic shake-off (Fig. 3A). We
also tested whether nocodazole inhibits the stimulation of p70
in resting cells upon addition of EGF. Pretreatment of cells for
30 min with different concentrations of nocodazole did not
significantly inhibit the EGF-induced activation of p70
(Fig. 3B). Thus, these control experiments show
that the low p70
activity measured in nocodazole-treated
cells in Fig. 2B is not an artifact of drug treatment.
Figure 3: Effect of nocodazole on S6 kinase activity. A, mitotic cells were collected by shake-off after treatment with (+) or without(-) nocodazole (see ``Materials and Methods''). Total S6 kinase activity was measured in extract supernatants prepared from mitotic cells and from cells that were left on the plates after the shake-off (non-mitotic cells). B, confluent cells were treated with (+) or without(-) different concentrations of nocodazole for 30 min. Then the cells were treated with or without 5 nM EGF for 20 min. Total S6 kinase activity was measured.
Additional control experiments were performed to determine whether
kinase activation might be due to withdrawal of nocodazole rather than
to a specific cell cycle change. Removal of nocodazole from cycling
cells, resting cells, or cells in S phase after 4 h of treatment did
not significantly increase p70 or
p42
/p44
activity (data not shown).
Furthermore, release of cells from a hydroxyurea-induced S phase arrest
did not lead to kinase activation (data not shown). Therefore, the
activation of p70
and p42
/p44
seen in Fig. 2B is probably not a general
phenomenon seen whenever cells are released from a cell cycle block.
To further pinpoint when during M or G p70
and p42
/44
become activated,
shorter time points after release from the metaphase block were
examined (Fig. 4A). Tubulin staining (red) and DAPI
staining of DNA (blue) were done to follow the cells through the
different stages of mitosis and to determine when cells entered
G
. The immunofluorescence pictures show that the
metaphase-arrested cells had condensed DNA but no mitotic spindles (Fig. 4A, t = 0 min). Fifteen min after
release from the nocodazole block the mitotic spindles had reformed (Fig. 4A). Thirty min after release the sister
chromatids started to separate and 15 min later the cells were in
anaphase. One hour after release the cells had gone through telophase
and cytokinesis and had entered G
.
Figure 4:
Activation of p70 and MAP kinases during M/G
. Nocodazole-arrested
cells were induced to enter G
(see ``Materials and
Methods''). A, at the indicated times after release,
cells were fixed with paraformaldehyde and microtubules were stained
with a monoclonal antibody against tubulin (red; see
``Materials and Methods''). DNA was stained with DAPI (blue). B, cell extract supernatants were prepared at
the indicated times and p70
(upper
panel) and MAP kinases (lower panel) were examined on
Western blots (see ``Materials and
Methods'').
Examination of
p70 on Western blots showed that a minor fraction of
p70
was phosphorylated during anaphase (Fig. 4B, t = 45 min), but most of the
enzyme became hyperphosphorylated after cells had gone through
cytokinesis and entered G
(Fig. 4B, t
= 60 min). In contrast to p70
, a substantial
fraction of p42
/p44
was already highly
phosphorylated during anaphase of mitosis (Fig. 4B, t = 45 min). Together, these analyses show that
p70
and p42
/p44
display
different kinetics of activation in cells progressing from M into
G
. p70
is activated very early in G
and remains active throughout G
, whereas
p42
/p44
are activated at the end of
mitosis and remain active during very early G
( Fig. 2and 4).
Figure 5:
Localization of S6 kinase during the cell
cycle. A, immunofluorescence of fibroblasts which were
synchronized in G and stimulated as described in the legend
to Fig. 1. At the indicated times cells were fixed and incubated
with p70
antibody (green) and DAPI (blue). Resting, t = 0 h; stimulated, t = 45 min; late
G
, t = 13 h; S, t = 17 h; G
, t = 21 h; M, t = 24 h. B, immunofluorescence of
fibroblasts which were released after nocodazole treatment as described
in the legend to Fig. 2.
We also examined the distribution of
p70/p85
in cells released from a nocodazole
block. As was seen in Fig. 5A, the kinase colocalized
with DNA during mitosis (Fig. 5B). In very early
G
, after cytokinesis had occurred and the DNA had
decondensed, the speckled S6 kinase pattern appeared in the nucleus (Fig. 5B, t = 2 h) and then disappeared
1 h later. Thus, p70
/p85
is localized in
the cytoplasm at all times during the cell cycle but becomes enriched
at certain locations in the nucleus during S/G
phase and
early G
in cells released from a metaphase block.
Figure 6:
Effect of serum withdrawal on S6 kinase
activity. A, fibroblasts were synchronized in G by
serum starvation as described under ``Materials and
Methods.'' At t = 0 h 20% FCS was added to induce
cell cycle entry. Synchronized or asynchronously cycling fibroblasts
received no further treatment (control), or at various times
were incubated for 10 min without FCS or for 10 min without FCS and
then 20 min with FCS. Total S6 kinase activity was determined in cell
extract supernatants. B, Western blot analysis of
p70
in cell extract supernatants prepared in A. Numbers refer to treatments in A.
Figure 7:
Regulation of mutant p70 during the cell cycle. A, structure of expressed S6
kinase molecules. Mutant S6 kinase contains three Ser
Asp and
one Thr
Glu changes in the carboxyl-terminal phosphorylation
sites. Both wild-type and mutant proteins have an HA epitope at the
amino terminus. B, S6 kinase activity in extract supernatants
from fibroblasts transfected with wild-type p70
(pMV7+ wt S6K), mutant p70
(pMV7+ mutant S6K), or the pMV7 vector alone (pMV7) was measured in HA immunoprecipitates (see
``Materials and Methods''). Mitotic cells (M) were
collected after nocodazole treatment as described under
``Materials and Methods'' and G
cells were
obtained 2 h after reseeding mitotic cells into drug-free medium.
Contact-inhibited cells were treated for 15 min without (G
) or with (EGF) 5 nM EGF.
Contact-inhibited cells were also pretreated for 15 min with either 400
nM wortmannin (EGF+ WM) or 100 nM
rapamycin (EGF+ Rapa) before addition of EGF. S phase
cells were collected 16 h after addition of mitogens to serum-starved
cells as described under ``Materials and Methods'' (S+ FCS) and were incubated for 10 min with DMEM in the
absence of FCS (S+ DMEM). The autoradiograph shows
incorporation of
P
into S6 during the kinase
assay. Similar results were obtained in several independent experiments
(data not shown). The
P
in S6 was quantitated
by scintillation counting; results from two experiments were averaged
and values for pMV7 were subtracted as background. For pMV7+ wt
S6K: 486 cpm (M), 1720 cpm (G
), 635 cpm (G
),
2519 cpm (EGF), 62 cpm (EGF+ WM), 76 cpm (EGF+ Rapa), 6948
cpm (S+ FCS) and 1910 cpm (S + DMEM). For pMV7+ mutant
S6K: 225 cpm (M), 469 cpm (G
), 175 cpm (G
), 482
cpm (EGF), 10 cpm (EGF + WM), 128 cpm (EGF+ Rapa), 855 cpm (S
+ FCS), and 154 cpm (S + DMEM). A portion (one-fifth volume)
of the supernatants that had been subjected to precipitation with HA
antibodies were then immunoprecipitated with antibodies to
p70
. Endogenous S6 kinase was assayed in the
p70
immunoprecipitates (see ``Material and
Methods'') and quantitated by scintillation counting. For
pMV7+ wt S6K: 2010 cpm (G
) and 10,350 cpm (EGF). For
pMV7+ mutant S6K: 5745 cpm (G
) and 12,525 cpm (EGF).
For pMV7: 4000 cpm (G
) and 12,540 cpm
(EGF).
If the four carboxyl-terminal
phosphorylation sites are responsible for regulating the activity of
p70 during the cell cycle, the mutant kinase should
display the same level of activity at all times. To test this
prediction, mitotic and G
populations of fibroblasts were
collected and the tagged wild-type and mutant p70
were
assayed in HA immunoprecipitates. As expected, the activity of
recombinant wild-type p70
was low in mitotic cells and
increased when cells entered G
(Fig. 7B).
However, the mutant enzyme also became more active as cells moved from
M phase into G
. No p70
activity was
immunoprecipitated from cells transfected with the empty vector (Fig. 7B). To further characterize the behavior of the
mutant p70
, we examined its ability to respond to
mitogens in G
cells. Confluent fibroblasts were treated
with or without EGF and the tagged kinases were assayed in HA
immunoprecipitates. Both the wild-type and mutant kinases were
activated upon addition of EGF (Fig. 7B). The degree of
EGF-induced activation of the HA-tagged kinases (2.8-4.0 fold; Fig. 7B) was significantly lower than that seen with
endogenous p70
in the parental cells (Fig. 3B). We therefore measured the activity of
endogenous p70
in the transfected cells by adding
p70
antibodies to supernatants that had been precleared
with HA antibody. Assay of these immunoprecipitates showed that
activation of the endogenous p70
in pMV7-transfected
cells was also reduced (2.2-5.1 fold; see legend to Fig. 7B).
Having established that the mutant
p70 could still be activated during M/G
and
G
/G
, we asked if the mutant enzyme was also
sensitive to negative regulators of the p70
pathway such
as wortmannin (27) and rapamycin(14) . Indeed,
treatment of transfected fibroblasts with wortmannin or rapamycin
completely abolished the EGF-induced activation of both mutant and
wild-type p70
(Fig. 7B). In addition,
similar to the results obtained with endogenous p70
in
the parental cells (Fig. 6), withdrawal of FCS from transfected
cells in S phase led to a rapid decline in S6 kinase activity of the
mutant protein (Fig. 7B). Thus, the apparently normal
regulation of the p70
mutant suggests that the
carboxyl-terminal phosphorylation sites alone do not regulate
p70
activity at the G
/G
boundary
or at other stages during the cell cycle.
Our data suggest that there is
a difference in the way that p70 and
p42
/p44
are activated in
G
/G
cells as compared to M/G
cells.
The kinases in G
are able to respond immediately to signals
generated by external growth factors. By contrast, the enzymes in
mitotic cells are maintained in an inactive state despite the presence
of abundant growth factors in the medium, implying the existence of a
negative regulatory mechanism. We have found that when
metaphase-arrested cells are released into serum-free medium, mitosis
is completed but p70
is not activated (data not shown).
Thus, activation of p70
at M/G
is triggered
by the presence of external growth factors and not by the completion of
mitosis per se. Completion of mitosis appears to suppress the
negative regulatory mechanism, thus creating an environment that is
permissive for kinase activation. This negative regulation of
p70
and p42
/p44
might be
mediated by a component of the cell cycle machinery such as a cyclin,
which accumulates until the end of M phase and is then rapidly
degraded.
p70 and p42
/p44
were activated more strongly during the
G
/G
transition (Fig. 1B) than
during M/G
(Fig. 2B). This difference is
probably linked to the differential protein synthesis requirement of
these cells. Quiescent cells contain fewer ribosomes and synthesize
proteins at a lower rate than cycling cells(41) . A sustained
increase in protein synthesis is required for resting cells to
synthesize new ribosomes and other proteins essential for entry into S
phase(42) , and as a result the G
phase is
approximately 4 h longer than G
of cycling cells. Since S6
phosphorylation enhances the synthesis of certain proteins involved in
translation(2) , it seems consistent that cells moving from a
quiescent state into S phase would require higher levels of S6 kinase
activity than M/G
cells. In addition, an essential function
of MAP kinases in G
might be to phosphorylate an inhibitory
subunit of translation initiation factor eIF-4E, thereby stimulating
the overall rate of protein synthesis in response to
mitogens(43) .
Similar to our results, Tamemoto et al.(44) found that p42/p44
activity is low in nocodazole-arrested Chinese hamster ovary
cells and that the enzymes become active upon entry into G
.
Furthermore, p42
/p44
were re-activated at
around M phase of the next cell cycle(44) . The interpretation
of these results was that p42
/p44
are
activated biphasically, first in G
and then in
G
/M before the nocodazole arrest point. However, loss of
synchrony by the end of the first cell cycle would make it impossible
to determine whether activation occurred in G
/M or
M/G
. It has been proposed that activation of MAP kinases
might be a one-time event involved in releasing cells from an arrested
state, rather than a recurring event required for progression through
each cell cycle(45) . This hypothesis is based mainly on
experiments done with oocytes and early embryos. However, MAP kinase
activation at M/G
in fibroblasts ( Fig. 2and Fig. 4) and Chinese hamster ovary cells (44) suggests
that the requirement for MAP kinase activity during G
might
be different in rapidly dividing embryonic cells and in established
cell lines.
Ribosomal protein
S6 is present in both the cytoplasm and nucleus, where it is found in
nucleoli and in association with chromatin(19) . The nucleolar
pool of S6 has been shown to become phosphorylated in response to
treatment of cells with phorbol esters(19) . Since protein
synthesis does not take place in the nucleus, the function of
phosphorylated S6 protein in this compartment remains obscure. Although
it has been shown that p85 activity in the nucleus is
required for entry into S phase(17) , it is not known whether
the essential function of this enzyme is to phosphorylate S6 or another
substrate.
We have shown that the behavior of
p70/p85
and p42
/p44
is cell cycle-regulated. Because the activities of these enzymes
are sensitive to changes in the growth factor supply, one might imagine
that the kinases are part of a sensory system that evaluates growth
conditions and makes the decision to exit or remain in the cell cycle.
A tightly controlled interplay between these signal transduction
molecules and the cell cycle machinery might be important for ensuring
proper cell growth and proliferation.