From the Molecular and Cellular Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037
Received for publication, January 15, 2003
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ABSTRACT |
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During mitosis, the cyclin-dependent
kinase, Cdc2, signals the inactivation of major anabolic processes such
as transcription, mRNA processing, translation, and ribosome
biogenesis, thereby providing energy needed for the radical and
energetically costly structural reorganization of the cell. This is
accomplished by phosphorylation and inactivation of several key
anabolic elements, including TFIIIB, TFIID, RNA polymerase II, poly(A)
polymerase, and translation elongation factor 1 During the mitotic phase of the cell cycle, the substructure of
mammalian cells is reorganized through a tightly regulated series of
events, which ultimately ensures that each of the two resulting
daughter cells receives the appropriate complement of genetic material.
During this period, anabolic processes in the cell are actively
repressed (1-3), serving to spare energy required for costly mitotic
events such as chromosome condensation, breakdown of the nuclear
envelope, and formation of the mitotic spindle. Transcription, mRNA
processing, and translation are among the metabolic activities to
undergo mitotic silencing. The collective inactivation of these
processes is due in large part to phosphorylation of key pathway
components by the cyclin-dependent kinase, Cdc2/Cdk1. A
number of mitotic Cdc2 targets involved in the silencing of gene
expression have been identified, including RNA polymerase II, TFIID,
TFIIIB (reviewed in Ref. 4), poly(A) polymerase (5), and
elongation factor 1 Among the most energetically expensive processes in cycling cells is
ribosome biogenesis, which, as a result of transcription, processing,
translation, and assembly of ribosomal proteins, has been calculated to
demand as much as 80% of the energy expenditure of a proliferating
cell (reviewed in Ref. 7). Cdc2 may act at multiple levels during
mitosis to repress ribosome biogenesis. Inhibition of rDNA
transcription is achieved by disruption of the RNA polymerase I complex
through Cdc2-mediated phosphorylation of TATA-binding protein (8), the
TATA-binding protein-associated factor TAFI110 (8), and
thyroid transcription factor-1 (8). Additionally, Cdc2 may also
suppress the translation of ribosomal protein-encoding mRNAs
through regulation of S6K11
(9).
Much of what is known of the function and regulation of S6K1 derives
from studies conducted during the transition from the quiescent state
(G0) to G1. As cells re-enter the cell cycle
from G0, S6K1 is coordinately phosphorylated on multiple
residues, leading to a robust stimulation of kinase activity. In
quiescent cells, it is postulated that S6K1 is rendered inactive by
virtue of an interaction between the putative autoinhibitory C-terminal tail and the catalytic pocket. This serves both to hinder the interaction of S6K1 with its substrates as well as to conceal internally situated sites of phosphorylation essential for maximal enzyme activity (reviewed in Ref. 10). In response to serum, up to six
S/TP sites (i.e. Ser411, Ser418,
Thr421, Ser424, Ser429, and
Thr447) are phosphorylated (11-13), disabling the
autoinhibitory domain. However, phosphorylation of S/TP sites within
the S6K1 autoinhibitory domain is not sufficient for stimulation of the
catalytic activity as deletion variants lacking the autoinhibitory
domain (and the six S/TP sites residing therein) are activated in
response to mitogens to an extent similar to the full-length protein
(13, 14). In addition, within the kinase extension, phosphorylation of
Thr389 increases the affinity of the requisite S6K1 kinase,
phosphoinositide-dependent kinase 1 (PDK1) (15). PDK1 then
phosphorylates Thr229 within the T-loop of the catalytic
domain (16, 17) inducing maximal kinase activity. A requirement for
phosphorylation of Ser371, an additional S/TP site removed
from the S/TP cluster within the autoinhibitory domain, for kinase
activity has been demonstrated (18), although its order in the
hierarchy of serum-stimulated phosphorylations is unclear. Several
candidate Thr389 kinases have been suggested, including the
mammalian target of rapamycin (mTOR) (19), PDK1 (20), and NEK6/7
(21), although the provenance of the Thr389 kinase has yet
to be unequivocally demonstrated.
The nature of the proline-directed kinase (or kinases) that catalyzes
the serum-stimulated, multisite phosphorylation of S/TP sites is also
unclear. In vitro, a number of proline-directed kinases have
been shown to phosphorylate S6K1, including ERK1/2, JNK, and Cdc2 (22).
Ser411 lies within a strong consensus for Cdc2
phosphorylation (K/RpSPR/PR/K/H (23)) and is phosphorylated during
mitosis when the activity of Cdc2 is augmented (9). Furthermore, by
anion-exchange chromatography, significant H1 kinase activity coeluted
with a proline-directed kinase activity toward a peptide substrate (p70
S6 kinase autoinhibitory pseudosubstrate) encompassing the S6K1
C terminus (22). The H1 kinase activity was Suc1-precipitable (22),
supporting the premise that the cofractionating S6K1 kinase was Cdc2.
The catalytic activity of Cdc2 is reliant on the formation of a
Cdc2·cyclin B complex, also known as maturation promoting factor. The
abundance of the Cdc2 protein is constant throughout the cell cycle
(24), whereas cyclin B accumulates progressively throughout interphase
until late in mitosis, at which point it is rapidly destroyed by the
anaphase promoting complex (reviewed in Refs. 25 and 26). The
destruction of cyclin B, therefore, serves as a mechanism of Cdc2
inactivation. Additionally, the Cdc2·cyclin B heterodimer is
stabilized by phosphorylation of Cdc2 at Thr161 and is
maintained inactive by dual phosphorylation of Thr14 and
Tyr15 (reviewed in Ref. 27). At the end of G2,
Cdc2 is dephosphorylated at Thr14 and Tyr15 by
the Cdc25 phosphatase, inducing the rapid activation of Cdc2 and
triggering entry into mitosis (reviewed in Ref. 28).
Numerous mitotic Cdc2 substrates have been identified, all of which are
phosphorylated at S/TP sites. The abundance of mitotic Cdc2 substrates
is illustrated by the appearance of numerous phospho-epitopes recognized by the MPM-2 antibody upon entry into mitosis (29), although
some of these epitopes may result from phosphorylation by kinases other
than Cdc2. The substrate specificity of Cdc2 shows an absolute
requirement for proline in the +1 position, a secondary requirement for
Arg or Lys at +3, and a preference for basic residues at +2 or +3
positions (23). Although peptide library screens of potential
Cdc2·cyclin B substrates predict that HHHKpSPRRR represents the
optimal sequence of Cdc2 phosphorylation (23), circumstances in which
the consensus has been relaxed are not uncommon (30). Furthermore, many
Cdc2 substrates possess multiple S/TP sites, not all of which conform
to the consensus sequence of phosphorylation, e.g. lamins
(31), Src (32), eIF4E binding protein 1 (33), and poly(A) polymerase
(5). In the case of S6K1, eight S/TP sites exist, one of which,
Ser411, lies within a strong consensus for Cdc2
phosphorylation (RpSPRR), whereas the contexts of the remaining seven
diverge somewhat (Fig. 1). Given the
abundance of S/TP sites within S6K1 and that Cdc2 phosphorylates the
consensus site, Ser411, in vitro, we postulated
that other non-consensus S/TP sites were likely to be regulated by Cdc2
during mitosis. Here, we present biochemical, pharmacological, and
genetic evidence that Cdc2 phosphorylates S6K1 at multiple S/TP sites
during mitosis. Furthermore, we demonstrate that Cdc2 is required for
mitotic inactivation S6K1 in FT210 cells.
Antibodies and Reagents--
For immunoblotting, anti-S6K1
antibodies were raised in rabbits immunized with a synthetic peptide
corresponding to amino acids 476-487 (RQPNSGPYKKQA) of rat S6K1. For
immunoprecipitation, anti-S6K1 antibodies were purchased from Santa
Cruz Biotechnology (catalog no. sc-230). The
anti-phospho-Ser371 antiserum has been described previously
(34) and was generously provided by John Blenis (Harvard University).
Antibodies to S6K1 phosphorylated at Ser411 (catalog no.
sc-8416) and to Cdc2 (catalog no. sc-54) were also from Santa Cruz
Biotechnology. Antibodies to S6K1 phosphorylated at Thr389
(catalog no. 9205) and Thr421/Ser424 (catalog
no. 9204), antibodies to Cdc2 phosphorylated at Tyr15
(catalog no. 9111), and antibodies to unphosphorylated RPS6 (catalog no. 2212) and RPS6 phosphorylated at
Ser240/Ser244 (catalog no. 2215) were purchased
from Cell Signaling Technology. BioMag magnetic beads were purchased
from Polysciences, Inc. Nocodazole, sodium orthovanadate, thymidine,
and aphidicolin were acquired from Sigma. Roscovitine, purvalanol A,
rapamycin, PD098059, U0126, SB202190, and wortmannin were purchased
from Calbiochem.
Cell Culture and Transient Transfection--
HeLa cells were
propagated in Dulbecco's modified Eagle's medium supplemented with
fetal calf serum (10%, v/v) and the antibiotics penicillin and
streptomycin at 37 °C. For cell synchronization, cells were arrested
at metaphase by nocodazole treatment (0.4 µg/ml) for 24 h.
Alternatively, cells were synchronized at the G1/S boundary
by sequential thymidine (2 mM) and aphidicolin (1 µg/ml)
blocks. Cells were subsequently released into the cell cycle by removal
of aphidicolin and addition of fresh medium for the times indicated.
The murine carcinoma cell line, FT210, carrying a temperature-sensitive
CDC2 allele, was kindly provided by Fumio Hanaoka (Osaka
University) and maintained in RPMI supplemented with fetal calf serum
(10%, v/v) and antibiotics at 32 °C. To induce Cdc2 deactivation,
FT210 cells were cultured at the non-permissive temperature of
39.5 °C for the times indicated.
HEK293T cells were propagated in Dulbecco's modified Eagle's medium
supplemented with fetal calf serum (10% v/v), penicillin, and
streptomycin at 37 °C. Cells were transfected with 20 µg of plasmid DNA in 100-mm dishes using the calcium phosphate method. Cells
were harvested 48 h post-transfection.
Cell Lysis and Immunoprecipitation--
Lysates were prepared
either from freshly isolated cells or from cell pellets frozen at the
time of harvest. Cells were extracted in lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40 (v/v), 150 mM NaCl, 1.5 mM EDTA, 1 µg/ml leupeptin, 1 mM Na3VO4, 500 µM
phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 mM NaF, 1 mM benzamidine, and 1 µg/ml
aprotinin. Lysates were rocked for 20 min at 4 °C on an end-over-end
rotator and then clarified by centrifugation at 15,000 × g at 4 °C for 25 min. The protein concentration of cell
lysates was determined using the Bio-Rad DC Protein Assay kit. Lysates
were either mixed with an equal volume of 2× sample buffer and heated
at 100 °C for 5 min or subjected to immunoprecipitation.
For immunoprecipitation, lysates were incubated for 2 h with
preformed antibody complexes immobilized on BioMag magnetic particles. S6K1 was immunoprecipitated with rabbit polyclonal S6K1 antibody (Santa
Cruz Biotechnology, catalog no. sc-230), Cdc2 with mouse monoclonal
anti-Cdc2 antibody (Santa Cruz Biotechnology, catalog no. sc-54), and
ectopically expressed HA-S6K1 with 12CA5 mouse monoclonal antibody.
Immune complexes were then washed three times with lysis buffer and
three times with high salt buffer (identical to lysis buffer except
NaCl concentration is 500 mM instead of 150 mM). Immune complexes were either resuspended in 1× sample buffer and heated at 100 °C for 5 min or subjected to in
vitro kinase assay.
Assay of S6 Kinase Activity--
The activity of S6K1 was
assayed exactly as outlined elsewhere (35). Briefly, anti-S6K1 immune
complexes were incubated with a peptide substrate (AKRRRLSSLRA), and
32P incorporation into the substrate was monitored by
liquid scintillation counting.
Assay of Cdc2 Kinase Activity--
The activity of Cdc2 was
determined in Cdc2 immunoprecipitates using either purified histone H1
or immunoprecipitated HA-S6K1 as substrate. Immune complexes were
washed as described above, washed once in reaction buffer (25 mM Tris-HCl, pH 7.4, 10 mM MgCl2),
and incubated at 30 °C in reaction buffer in the presence of 2 µg
of purified histone H1 (Calbiochem, catalog no. 382150) and 1 µCi of
[ Cell Cycle Analysis--
Cell cycle analysis was performed by
flow cytometry. Briefly, cells were trypsinized, washed in
phosphate-buffered saline, fixed in ice-cold 70% ethanol, and treated
simultaneously with propidium iodide (40 µg/ml) and RNase A (200 µg/ml) for 1 h at 37 °C. Cells were subsequently analyzed on
a FACScan with CellQuest software.
Although the specific activity of S6K1 is reduced in
nocodazole-arrested Cos7 cells compared with cells in interphase (9), a
similar treatment has no apparent effect in Swiss 3T3 fibroblasts (36).
Furthermore, phosphorylation of the consensus Cdc2 S/TP site,
Ser411, is induced both in nocodazole-arrested Cos7 cells
and in primary human T cells during G2/M (9). We therefore
sought to determine whether S6K1 prepared from nocodazole-arrested HeLa
cells displayed reduced kinase activity and whether or not this was
associated with changes in site-specific phosphorylation. Initially,
FACS analysis was performed on growing HeLa cells incubated for
increasing periods with nocodazole (Fig.
2A). These results indicate
that 24 h of nocodazole exposure induced a near-complete M phase
arrest. We therefore chose a 24 h incubation for all subsequent
nocodazole treatments. This M phase arrest was associated with a
prominent activation of Cdc2 as indicated by dephosphorylation of
Tyr15 (Fig. 2B) and by an increase in H1 kinase
activity detected in anti-Cdc2 immunoprecipitates (Fig.
2C).
. We report here that
ribosomal S6 kinase 1 (S6K1), a protein kinase linked to the
translation of ribosomal protein mRNAs, is also subject to
regulation by Cdc2 in mitosis. In mitotic HeLa cells, when the activity
of Cdc2 is high, S6K1 is phosphorylated at multiple Ser/Thr, Pro (S/TP)
sites, including Ser371, Ser411,
Thr421, and Ser424. Concomitant with this, the
phosphorylation of the hydrophobic motif site, Thr389, is
reduced resulting in a decrease in the specific activity of S6K1. The
mitotic S/TP phosphorylation sites are readily phosphorylated by
Cdc2·cyclin B in vitro. These proline-directed
phosphorylations are sensitive to chemical inhibitors of Cdc2 but not
to inhibitors of mammalian target of rapamycin, phosphatidylinositol
3-kinase, MEK1/2, or p38. In murine FT210 cells arrested in mitosis,
conditional inactivation of Cdc2 reduces phosphorylation of S6K1 at
S/TP sites while simultaneously increasing phosphorylation of
Thr389 and of the S6K1 substrate, RPS6. A physical
interaction exists between Cdc2 and S6K1, and this interaction is
enhanced in mitotic cells. These results suggest that Cdc2 provides a
signal that triggers inactivation of S6K1 in mitosis, presumably
serving to spare energy for costly mitotic processes at the expense of
ribosomal protein synthesis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
(6).
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Fig. 1.
Position of S/TP phosphorylation sites of
S6K1. A, depiction of the full-length S6K1 molecule.
Each of the four modular domains is highlighted with eight S/TP
phosphorylation sites displayed above the protein and three non-S/TP
sites displayed below the protein. The kinases shown to phosphorylate
S6K1 in vitro at individual sites are shown. B,
primary amino acid sequence of S6K1 comprising all S/TP sites and the
amino acid context in which they lie. The amino acid position is
indicated above the sequence. The underlined sequence
represents the optimal phosphorylation consensus for Cdc2.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP. Phosphorylated histone H1 or HA-S6K1 was
analyzed by SDS-PAGE and autoradiography.
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ABSTRACT
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DISCUSSION
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Fig. 2.
Mitotic arrest induces S6K1 inactivation
despite increased S/TP-specific phosphorylation. A,
HeLa cells were incubated with nocodazole (0.4 µg/ml) for the
times indicated. Histograms show cell cycle distribution at each time
point. Whole cell lysates (WCL) were prepared from HeLa
extracts of asynchronous populations (interphase, I) or
after 24-h incubation with nocodazole (mitotic arrest, M).
B, HeLa lysates were separated by SDS-PAGE then
immunoblotted with antibodies specific for Cdc2 or Cdc2 phosphorylated
on Tyr15. C, Cdc2 was immunoprecipitated from
HeLa extracts and incubated in a kinase reaction using purified histone
H1 as substrate. The kinase reactions were separated by SDS-PAGE,
transferred to nitrocellulose membranes, and then subjected to
auto- radiography. A representative autoradiogram is presented.
D, HeLa lysates were immunoblotted with antibodies specific
for S6K1 or S6K1 phosphorylated on Ser371,
Ser411, Thr421/Ser424, or
Thr389. E, S6K1 was immunoprecipitated from HeLa
extracts and incubated with a synthetic peptide substrate corresponding
to the region of phosphorylation of RPS6. The reactions were spotted
onto filters and counted by liquid scintillation counting. The data are
representative of five independent experiments. F, HA-S6K1
was transiently expressed in and purified from asynchronous 293T cells
and incubated with purified, baculovirus-expressed Cdc2·cyclin
B in a Cdc2 kinase reaction. The reactions were separated by SDS-PAGE,
transferred to polyvinylidene difluoride membranes, and either
subjected to autoradiography or immunoblotted with
anti-phosphopeptide antisera as indicated. The data are
representative of three independent experiments.
We observed an interesting profile of phosphorylation for mitotic S6K1. Using anti-phosphopeptide antibodies, four S/TP sites, Ser371, Ser411, Thr421, and Ser424, showed reproducible increases in phosphorylation in response to nocodazole, although to differing extents (Fig. 2D). The fold increases in site-specific, mitotic phosphorylation were as follows: Ser371, ~50%; Ser411, ~100%; and Thr421 and Ser424, ~1000%. This was in stark contrast to Thr389, which showed substantial dephosphorylation upon mitotic arrest (Fig. 2D). Because stimulation of the kinase activity of S6K1 is absolutely reliant upon phosphorylation of Thr389, the reduction in S6 kinase activity measured in anti-S6K1 immunoprecipitates prepared from mitotic cells was consistent (Fig. 2E). It is noteworthy, however, that multisite phosphorylation of S/TP sites is predicted to disengage the autoinhibitory function of the C-terminal tail, thereby facilitating (and indeed being associated with) the activation of S6K1. These findings, therefore, are suggestive of a novel function of mitotic S/TP phosphorylation distinct from the mitogen-stimulated activation of S6K1.
Previous studies have established that S6K1 is a substrate of Cdc2·cyclin B in vitro, particularly at Ser411 (9). Given the presence of multiple, neighboring S/TP sites within the S6K1 protein, and the fact that these sites are phosphorylated in mitotic cells, we reasoned that these sites may be substrates for Cdc2·cyclin B as well. N-terminal HA-tagged S6K1 was transiently expressed in HEK293T cells and immunoprecipitated from asynchronous cells with anti-HA antibody. Active Cdc2·cyclin B complexes were expressed in and purified from baculovirus-infected Sf9 cells and used to phosphorylate immunopurified HA-S6K1 in vitro. Of the S/TP sites evaluated, Ser411, Thr421, and Ser424 were heavily phosphorylated, whereas Ser371 was only modestly phosphorylated. The non-proline-directed hydrophobic motif site, Thr389, was not phosphorylated in the reaction (Fig. 2F). Thus, phosphorylation of S6K1 by Cdc2·cyclin B is specific for Ser/Thr in S/TP motifs, although both consensus and non-consensus sites are efficiently phosphorylated.
To confirm that the observed effects on S6K1 were due to mitotic arrest
rather than nonspecific effects secondary to microtubule destabilization, we monitored the pattern of site-specific
phosphorylation of S6K1 throughout the HeLa cell cycle by
immunoblotting with anti-phosphopeptide antibodies. Cells were
synchronized at the G1/S boundary by sequential
thymidine/aphidicolin blocks and then released into the cell cycle.
FACS analysis revealed that synchronization of these cells was achieved
and maintained over the entire cell cycle (Fig.
3A). Generally, from early S
phase to G2/M (0-9 h), the phosphorylation of S6K1
at the S/TP sites, Ser371, Thr421, and
Ser424, rose steadily (Fig. 3B). Upon entry into
the new cycle, these sites were dephosphorylated, and then subsequently
their phosphorylation increased as the latter half of the subsequent
phase approached (13-27 h). The phosphorylation of ribosomal protein
S6 (RPS6), a natural S6K1 substrate, was elevated from early S phase (3 h) to G2/M (9 h) then returned to basal levels as cells
progressed into the new cycle (Fig. 3B, histogram). It is
important to point out that, although populations are enriched in the
G2/M phase of the cell cycle at 9 h after release from
the G1/S block, FACS analysis cannot distinguish between
G2 and M phase cells. Dephosphorylation of Cdc2 at
Tyr15 by Cdc25 is an event that triggers the activation of
Cdc2 and entry into mitosis (reviewed in Ref. 28). Hence,
Tyr15 phosphorylation and dephosphorylation is a reliable
marker of G2 and M phases, respectively. In view of this,
the presence of Tyr15 phosphorylation at 9 h (Fig.
3B) indicates that most of these cells had yet to enter
mitosis and thus were synchronized in G2. Furthermore,
although by 13 h of release, cells had accumulated in
G1, a small percentage remained in the G2/M
phase; these are likely to have been in M phase rather than
G2 (see below).
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To precisely monitor the site-specific phosphorylation of S6K1 during
mitosis, we performed a narrower time course corresponding to the
interval between 8.5 and 15 h after release from G1/S
block. In these experiments, FACS analysis was carried out in
conjunction with a visual assay of Hoescht-stained HeLa cells to
identify those times at which the percentage of mitotic cells was
greatest. The cell cycle distribution at each time point is shown in
Fig. 4A. Based on this
analysis, the period of 8.5-10 h comprised the end of G2,
whereas that of 13.5-15 h represented the beginning of G1.
The period from 10.5-12.5 h displayed the greatest enrichment in
mitotic cells as determined by Hoescht staining. It is important to
point out that only ~50% of Hoescht-stained cells were discernibly mitotic at 11.5 h, although this time point displayed the greatest mitotic index (data not shown). The C-terminal S/TP sites of S6K1, Ser411, Thr421, and Ser424, all
displayed peak phosphorylation during mitosis (Fig. 4B). Conversely, RPS6 was increasingly dephosphorylated as cells entered mitosis, concomitant with dephosphorylation of Cdc2 at
Tyr15 (Fig. 4, compare B-D).
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Given that Cdc2 phosphorylates S6K1 at multiple S/TP sites in
vitro (Ref. 9 and Fig. 2F) and that Cdc2
phosphorylation of non-consensus S/TP sites in other Cdc2 substrates is
not uncommon (5), we reasoned that the non-consensus S/TP sites found
in S6K1 (e.g. Ser371, Thr421, and
Ser424) may also be under Cdc2 control in vivo.
It would then be reasonable to predict that pharmacological inhibition
of Cdc2 during mitosis would block the appearance of phospho-epitopes
at putative consensus and non-consensus Cdc2 sites. To test this
hypothesis, HeLa cells were arrested in mitosis with nocodazole
followed by the addition of various phosphatase and kinase inhibitors.
The addition of sodium orthovanadate, a tyrosine phosphatase inhibitor,
has been classically used to inhibit tyrosine dephosphorylation of Cdc2 by the dual-specificity phosphatase, Cdc25 (37), thereby maintaining Cdc2 inactivity. Because the Thr14 and Tyr15
kinases, Wee1 and Myt1, remain active (or, in fact, may be activated as
a result of inhibition of Cdc2), Cdc2 is rephosphorylated after vanadate-induced inhibition of Cdc25. Addition of this inhibitor resulted in dephosphorylation of S6K1 at both consensus
(Ser411) and non-consensus (Thr421 and
Ser424) Cdc2 sites (Fig.
5A). Similar results were
obtained with the cyclin-dependent kinase inhibitors,
roscovitine and purvalanol A, insofar as S/TP site phosphorylation is
concerned. Proline-directed phosphorylation of mitotic S6K1 was both
rapamycin-resistant (Fig. 5A) and wortmannin-resistant (Fig.
5B), precluding the possibility that either mTOR or
phosphatidylinositol 3-kinase controls the S/TP-specific
phosphorylation of S6K1 during mitosis. However, the residual
phosphorylation of Thr389, a rapamycin-sensitive, non-S/TP
phosphorylation site within the S6K1 kinase extension, which persists
in mitotic cells, was further dephosphorylated in response to rapamycin
(data not shown). This indicates that mTOR retains the ability to
signal to S6K1 during mitosis. Other proline-directed kinases
reportedly active in mitotic cells (38, 39) were also evaluated as
candidate regulators of S6K1. However, inhibitors of the MEK/ERK
pathway, U0126 and PD098059 (Fig. 5A), and of the
mitogen-activated protein kinase, p38, SB202190 (Fig. 5B),
had little effect on the phosphorylation of S/TP sites. Collectively,
the results of these inhibitor studies provide compelling evidence that
Cdc2 is likely to be the primary mitotic S6K1 kinase.
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If Cdc2 represents the major mitotic kinase regulating S/TP site
phosphorylation of S6K1, then genetic inactivation of Cdc2 should
prevent such phosphorylation. In mammalian cells, a
temperature-sensitive CDC2 allele has been identified at the
genomic locus of the FT210 murine carcinoma cell line (40). The
CDC2 gene product in these cells harbors two point mutations
(I54V and P272S) that promote its rapid inactivation and degradation at
the restrictive temperature of 39 °C (40). As a result, FT210 cells
are unable to enter mitosis and accumulate in G2 when
cultured at 39.5 °C (Ref. 40 and Fig.
6A). To test whether active
Cdc2 kinase is necessary for mitotic S/TP-specific phosphorylation of
S6K1, mitotic arrest was induced with nocodazole at the permissive
temperature (32 °C), and cells were then transferred to the
restrictive temperature to inactivate Cdc2. Based on the results of
FACS analysis, this treatment induces a complete M phase arrest (Fig.
6A). We then switched the nocodazole-arrested cells to the
restrictive temperature for increasing lengths of time and assayed
site-specific phosphorylation of S6K1 by immunoblotting. Consistent
with a critical role for Cdc2 in the regulation of S/TP-specific
phosphorylation, S6K1 was increasingly dephosphorylated at
Ser411, Thr421, and Ser424, when
cells were incubated at the non-permissive temperature (Fig.
6B). These changes temporally paralleled the loss of Cdc2 protein observed at the restrictive temperature. Interestingly, after
6 h at the restrictive temperature, when the abundance of Cdc2
protein was substantially reduced, RPS6 was rephosphorylated, as was
S6K1 at Thr389 (Fig. 6B). These findings
demonstrate not only that Cdc2 is necessary for S/TP-specific
phosphorylation of S6K1 during mitosis but also that Cdc2 is necessary
for inhibition of S6K1 in mitotic cells.
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It has been reported that a significant H1 kinase activity copurifies
with an S/TP kinase activity specific for S6K1 C terminus (amino acids
400-436; also called p70 S6 kinase autoinhibitory pseudosubstrate peptide) prepared from H4 hepatoma cells (22). Because
Cdc2-mediated regulation of S6K1 is likely to reflect direct
phosphorylation of S/TP sites most notably during M phase, we reasoned
that a complex of S6K1 and Cdc2 may be detectable by
immunoprecipitation. We were able to detect Cdc2 in anti-S6K1 immunoprecipitates from interphase HeLa extracts (Fig.
7). Importantly, the formation of the
S6K1·Cdc2 complex was enhanced ~3-fold during mitosis We have been
unable to detect site-specific differences in phosphorylation of S6K1
between that pool bound to Cdc2 and that found in a whole cell extract
(data not shown) suggesting that phosphorylation of S6K1 by Cdc2 does
not affect the association of the two proteins.
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DISCUSSION |
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In the present study, we have demonstrated that S6K1 is phosphorylated on both consensus and non-consensus S/TP residues in vitro by Cdc2·cyclin B and in vivo in a mitosis-specific fashion. Mitotic phosphorylation of S6K1 is associated with its inactivation as evidenced by decreased specific kinase activity and dephosphorylation of its substrate, RPS6. We have provided both pharmacological and genetic evidence suggesting that Cdc2 represents the major mitotic regulator of S6K1. Our results demonstrate that Cdc2 is required for mitotic phosphorylation of S/TP sites as well as mitotic inhibition of S6K1. Finally, Cdc2 and S6K1 form an immunoprecipitable complex whose stability is enhanced in mitotic cells.
An essential role of S6K1 in the G1/S phase transition has been inferred from two lines of evidence. First, Lane et al. (41) demonstrated that microinjection of neutralizing anti-S6K1 antibodies prevented serum-induced S phase entry in fibroblasts. Second, rapamycin, which rapidly inactivates S6K1 (42-46), also delays or inhibits cell cycle progression, specifically in G1 (42, 43, 47). In view of this body of evidence, much effort has been focused on understanding cell cycle regulation of S6K1 in the context of the G0/G1 transition. The addition of serum to quiescent cells induces phosphorylation of multiple S/TP sites within the autoinhibitory tail as well as within the kinase domain at Thr229 and Thr389. Collectively, this series of events leads to a rapid increase in the kinase activity of S6K1 (48). Using synchronous populations of cells released from G1/S arrest, we have observed that phosphorylation of S6K1 at S/TP sites and of RPS6 is enhanced during late phases of the cell cycle. However, upon mitotic entry, S/TP site phosphorylation of S6K1 is further stimulated, whereas RPS6 is dephosphorylated. This indicates that, despite enhancd phosphorylation of S6K1 at multiple S/TP sites, the S6K1/RPS6 pathway is inactivated during mitosis. In synchronized HeLa cells, the incorporation of ribosomes into larger polysomes is greatest during G2 (49), consistent with the G2 phase representing a period of high translational activity. We found that the phosphorylation of RPS6 was not significantly stimulated as cells entered G1 from M phase. Similar results were obtained from HeLa cells released from nocodazole arrest (data not shown). Collectively, these data argue that, in the absence of G0 arrest, the machinery responsible for the translation of ribosomal protein mRNAs is engaged in the latter half of the cell cycle.
The findings that mitotic phosphorylation of the S/TP sites, Ser411, Thr421, and Ser424, is abrogated by Cdc2 inhibitors and is reduced in cells in which Cdc2 has been conditionally inactivated suggests that Cdc2 is likely to be the primary mitotic S6K1 regulator. This hypothesis is further supported by the fact that Ser411 (Ref. 9 and Fig. 2F) as well as Ser371, Thr421, and Ser424 (Fig. 2A) are directly phosphorylated by Cdc2 in vitro and that the association of Cdc2 and S6K1 is induced in mitotic cells (Fig. 7). Eight S/TP sites are included in the S6K1 protein: two in the kinase extension (i.e. Thr367 and Ser371) and six in the C-terminal autoinhibitory tail (i.e. Ser411, Ser418, Thr421, Ser424, Ser429, and Thr447). It should be noted that, although the phosphorylation of S6K1 at Thr367, Ser418, Ser429, and Thr447 were not directly measured in this study, these residues might represent additional sites phosphorylated by Cdc2 in mitotic cells.
Among the S/TP sites, only Ser411 conforms to the consensus for Cdc2 phosphorylation (23, 30). Cdc2 substrates often possess a combination of consensus and non-consensus S/TP sites. The consensus sites of one such substrate, poly(A) polymerase, respond to lower levels of Cdc2 kinase activity than do non-consensus sites, although phosphorylation of all sites is necessary for mitosis-specific regulation of the catalytic activity of poly(A) polymerase (5). This led to the interesting postulation that particular Cdc2 substrates may, by virtue of the presence of particular combinations of consensus and non-consensus sites, respond to varying levels of Cdc2 activity. In the case of S6K1, we observed increased phosphorylation of the Cdc2 consensus site, Ser411, during G2, whereas phosphorylation of Thr421 and Ser424 was increased only upon entry into mitosis (Fig. 4B). Therefore, Cdc2-mediated regulation of S6K1 may rely upon the cumulative effect of multiple S/TP phosphorylations that can only be achieved during mitosis, when the activity of Cdc2 is highest. It should be emphasized that phosphorylation of S6K1 by Cdc2 may serve some regulatory function without influencing S6K1 activity (see below). The increase in phosphorylation of S6K1 at Ser411 may in fact derive from phosphorylation by a Cdk·cyclin A complex, such as Cdc2·cyclin A, which is active in G2. It is likely, however, that Cdc2·cyclin B phosphorylates S6K1 at the G2/M transition and throughout M phase.
The observation that loss of the Cdc2 protein in mitotically arrested cells temporally coincides with increased phosphorylation of RPS6 (Fig. 6B) implies that one function of active Cdc2 is to signal mitotic inactivation of S6K1. Initially this might seem paradoxical, because phosphorylation of multiple S/TP sites within the C-terminal tail is predicted to disengage the autoinhibitory mechanism, thereby "priming" S6K1 for further activation. In light of the observation that multisite, proline-directed phosphorylation is reduced upon entry into G1 (Fig. 4, A and B), it is unlikely that mitotic phosphorylation would serve to prime S6K1 for activation in G1, especially because RPS6 is not phosphorylated during this period. What purpose might be served by multisite S/TP phosphorylation of S6K1 in mitotic cells? Potentially, these phosphorylations could alter the binding preference of S6K1 for mitosis-specific interaction partners. In fact, phosphorylation of the Cdc2 consensus site, Ser411, is predicted to create an optimal binding surface for the peptidyl-prolyl cis/trans isomerase, Pin1 (50), which has been shown to interact with S6K1 in a mitosis-specific fashion (50). Given that the S/TP-rich C-terminal tail is postulated to regulate S6K1 activity via a phosphorylation-dependent change in conformation, Pin1 may play an active role in this process during mitosis. Pin1 has been shown to facilitate dephosphorylation of S/TP sites by PP2A (51, 52), potentially explaining the rapid dephosphorylation of S6K1 at these sites at the point of mitotic exit. Studies are currently in progress to address these possibilities.
The data presented in Figs. 2 and 6 suggest that Cdc2 signals the
inactivation of S6K1 in mitotic cells by inducing dephosphorylation of
Thr389. Culturing mitotically arrested FT210 cells at the
restrictive temperature results in rephosphorylation of
Thr389, an effect that tightly parallels disappearance of
the Cdc2 protein (Fig. 6). Cdc2 is therefore necessary for mitotic
regulation of S6K1 by reducing the extent to which Thr389
is phosphorylated. Given that Cdc2 is highly active in mitotic cells
and that Thr389 does not fall within a preferred S/TP
context for phosphorylation by Cdc2, the regulation of
Thr389 phosphorylation by Cdc2 is likely to be indirect.
Naturally, the reduction in phosphorylation of Thr389 could
derive from inhibition of the Thr389 kinase or stimulation
of the Thr389 phosphatase. Reconciliation of this issue is
complicated, however, by the fact that neither the Thr389
kinase nor the Thr389 phosphatase has been unequivocally
described. Nevertheless, mTOR represents an attractive intermediary
target in the regulation of Thr389 phosphorylation by Cdc2.
The phosphorylation of Thr389 is sensitive to the mTOR
inhibitor, rapamycin, which leads to the rapid inactivation of S6K1 in
the presence of the compound (53). In vitro studies have
demonstrated that mTOR can phosphorylate Thr389 (19, 54);
however, several rapamycin-resistant S6K1 constructs have now been
described in which Thr389 is phosphorylated under
mTOR-inactivating conditions (e.g. rapamycin treatment
(55-57) and amino acid withdrawal (55)). Thus, the most plausible
explanation of these observations is that mTOR indirectly controls
Thr389 phosphorylation of S6K1. It has been reported
elsewhere that nocodazole arrest stimulates the kinase activity of mTOR
in DOHH2 cells (58). Nevertheless, we cannot exclude the possibility that mTOR is inactivated in mitotic HeLa and FT210 cells. Collectively, our data suggest a bimodal regulation of S6K1 by Cdc2 (Fig.
8): in mitotic cells, Cdc2 directly
phosphorylates S6K1 at multiple S/TP motifs and concomitantly inhibits
the pathway affecting the phosphorylation of Thr389. This
latter effect is indirect and accounts for the reduced activity of
mitotic S6K1 compared with interphase S6K1.
|
The importance of S6K1 in cell cycle progression is underscored by the
observation that the S6K1 gene is amplified or overactive in
primary breast cancers (59, 60), ovarian cancers (61), and in
phosphatase and tensin homolog deleted on chromosome 10-null tumors
(62). Additionally, not only is ribosome biogenesis a strong prognostic
indicator of tumorigenicity (63), but significant, positive
correlations have been established between the up-regulation of
ribosomal protein-encoding mRNAs and the growth rate of transformed cells (64). Additionally, overexpression of wild-type S6K1, but not an
inactive variant, in NIH3T3 cells appears to interfere with cytokinesis
as evidenced by the multinucleation and polyploidization of transfected
cells (65). This suggests that failure to inactivate S6K1 during
mitosis would deleteriously affect the integrity of the genome as well
as the growth characteristics of the cell.
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ACKNOWLEDGEMENTS |
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We thank Angela Romanelli and John Blenis for critical reading of the manuscript and for the anti-phospho-Ser371 antibody used in this study. We also thank Fumio Hanaoka for supplying the FT210 cells.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Services Grants CA82683 and CA14195.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by NIH Grant T32-CA09523-18 and a Pioneer fellowship.
§ Supported by Jane Coffin Childs Memorial Fund for Medical Research Grant 61-1210.
¶ A Frank and Else Schilling American Cancer Society Research Professor. To whom correspondence should be addressed: Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-453-4100 (ext. 1385); Fax: 858-457-4765; E-mail: hunter@salk.edu.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M300435200
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ABBREVIATIONS |
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The abbreviations used are: S6K1, ribosomal S6 kinase 1; PDK1, phosphoinositide-dependent kinase 1; S/TP, Ser/Thr, Pro sites; mTOR, mammalian target of rapamycin; HA, hemagglutinin; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; JNK, c-Jun N-terminal kinase; FACS, fluorescence-activated cell sorting.
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