From INSERM EMI-U 9928, Centre Hospitalier Universitaire Angers, 4 rue Larrey, 49033 Angers Cedex, France
Received for publication, January 29, 2001, and in revised form, February 8, 2001
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ABSTRACT |
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STAT3 transcription factors are cytoplasmic
proteins that induce gene activation in response to cytokine receptor
stimulation. Following tyrosine phosphorylation, STAT3 proteins
dimerize, translocate into the nucleus, and activate specific target
genes. Activation is transient, and down-regulation of STAT3 signaling
occurs within a few hours. In this study, we show that cyclin D1
inhibits STAT3 activation. In co-immunoprecipitation and pull-down
assays, cyclin D1 was found to associate with the activation domain of
STAT3 upon interleukin-6 stimulation. Overexpression of cyclin D1
inhibited transcriptional activation by STAT3 proteins. This effect was not shared by cyclin E, was independent of association with
Cdk4, and was unaffected by inhibitors of Cdk4. Whereas cyclin
D1 had no effect on the steady-state level of STAT3 proteins in the
cytoplasm, it was found to reduce the STAT3 nuclear level in HepG2
cells. These results suggest a model by which cyclin D1 is part of a feedback network controlling the down-regulation of STAT3
activity and highlight a new activity for this cell cycle regulatory protein.
The JAK1/STAT pathways
are activated by various growth factors and cytokines such as epidermal
growth factor and interleukin-6 (IL-6). Binding of these cytokines to
their receptors activates the JAK tyrosine kinases, followed by
tyrosine phosphorylation of the receptors. This leads to activation and
homo- or heterodimerization of the STAT1/3 transcription factors,
translocation into the nucleus, and activation of target genes (1, 2).
STAT3 activation is often associated with cell growth or
transformation, and disruption of the stat3 gene causes
embryonic lethality around remove embryonic day 7.5, confirming a role
for STAT3 in cell survival and proliferation in embryonic development
(3). Recent experiments indicate that STAT3 transcription factors
induce cell transformation and can be considered as oncogenes (4, 5).
Tumor-derived cell lines or samples from human cancer contain very
frequently activated forms of STAT3, and all src-transformed
cell lines exhibit activated STAT3 (4, 6-9). Coexpression of a
dominant-negative form of STAT3 is sufficient to block cell
transformation by Src (7, 9). Inhibition of STAT3 transcriptional
activity can decrease Bcl-xL expression and induces
apoptosis in U266 cells (10) as well as in cultures of primary human
myeloma cells (4). Finally, STAT3 is also required for the
IL-6-mediated activation of the c-myc gene (11) and for the
expression of cyclin D1 (5), two key components in the regulation of
cell cycle progression from G1 to S phase.
One of the important questions to be resolved is what molecular basis
governs the activation of cell cycle progression by STAT3 transcription
factors and how these proteins are inactivated. Cell cycle activation
is coordinated by D-type cyclins, which are rate-limiting
and essential for the progression through the G1 phase of
the cell cycle (12). D-type cyclins bind to and activate the
cyclin-dependent kinases Cdk4 and Cdk6 (13). This binding
allows for efficient phosphorylation of the critical downstream target
of cyclin D, the retinoblastoma protein Rb. In the G1 phase of the cell cycle, Rb is hypophosphorylated and binds to the E2F family
of cell cycle transcription factors (14). This Rb·E2F complex forms
an active transcriptional repressor at the promoter of cell cycle genes
that is responsible for growth suppression (15). The binding of Rb to
E2F and the ability to repress transcription are modulated by the state
of phosphorylation of Rb catalyzed by cyclin-dependent
kinases. The complete phosphorylation of Rb by cyclin D1/Cdk4 and
cyclin E/Cdk2 is necessary for its inactivation, resulting in the
release of Rb-associated E2F and the activation of S phase genes
(16-18).
In the past 3 years, several observations have raised the possibility
that cyclin D1 has roles at other stages of the cell cycle and that
these new activities are unrelated to its function as a Cdk regulatory
subunit (19). Cyclin D1 can activate estrogen receptor transcription
through a direct interaction with the ligand-binding domain of the
receptor (20, 21). This effect is independent of Cdk4 and is explained
by the recruitment of the SRC1a family of coactivators by cyclin D1
(22). In line with these observations, cyclin D1 inhibits
transcriptional activation by the v-myb oncogene, and this
repression is also independent of a complex formation with Cdk4 (23,
24). The transcriptional activity of another Myb-like protein, DMP1, is
also antagonized by D-type cyclins through a Cdk-independent mechanism
(25). Finally, cyclin D1 has been shown to repress muscle
differentiation and MyoD-mediated transcription in part through an
Rb-independent mechanism (26, 27). Taken together, these studies
establish a new Cdk-independent role of cyclin D1 as a transcriptional
regulator (19).
STAT proteins activate the expression of the cyclin D1 gene through the
recognition of a conserved element in the promoter of the gene (5, 28).
As one of the target genes activated by the STAT cascade, cyclin D1 is
probably an important mediator of the STAT-dependent growth
of hematopoietic cells. However, the exact role of this protein in this
signaling pathway remains to be determined. In this study, we show that
following IL-6 stimulation, cyclin D1 interacts with STAT3 and inhibits
its transcriptional activity. This effect is not shared by cyclin E, is
independent of Cdk4, and is related to reduced STAT3 nuclear expression
in HepG2 cells. These results reveal an unexpected relationship between cyclin D1 and STAT3 proteins and led us to propose that cyclin D1 is
part of a feedback network controlling the down-regulation of STAT3 activity.
Cell Culture and Reagents--
Cell lines obtained from the
American Type Culture Collection (Manassas, VA) were grown in RPMI 1640 medium supplemented with 10% fetal calf serum or in RPMI 1640 medium
supplemented with 10% fetal calf serum and 1 ng/ml
granulocyte/macrophage colony-stimulating factor for TF1 cells.
Purified recombinant cytokines were obtained from Dr. K. Turner
(Genetics Institute, Boston, MA). Vectors expressing cyclin D1
(pRC/cmv), p16INK4 (pcDNA3), and cyclin D1-KE (pRC/cmv) were
kind gifts of Drs. P. Whyte and R. Bernards, respectively. The
Gal4-STAT3 and Gal4-VP16 fusion proteins and the
Gal4-luciferase reporter gene system were kindly provided by
Drs. D. E. Levy and F. Gouilleux. Plasmid expressing gp130 has
been described previously (29). Polyclonal antibodies recognizing STAT3
(C20) and cyclin D1 (H-295) were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). Polyclonal antibodies directed against phospho-STAT3 Tyr705 and phospho-STAT3 Ser727 were obtained
from New England Biolabs Inc. (Beverly, MA).
Plasmid Constructs--
Expression vectors were used as
templates for polymerase chain reaction amplification of various
portions of cyclin D1 and STAT3. GST-cyclin D1 fusion constructs were
prepared in pGEX-2T using oligonucleotide primers containing
BamHI and EcoRI restriction sites corresponding
to amino acids 1-295 of cyclin D1. Histidine-tagged STAT3 fusion
constructs were prepared in pET15b using oligonucleotide primers
containing BamHI restriction sites corresponding to amino acids 716-770 of STAT3.2
Transient Transfections and Preparation of Nuclear
Extracts--
Transfection experiments were done using the calcium
phosphate precipitation method and were repeated at least five times. COS cells were transfected following the DEAE-dextran method. The
amount of transfected DNA was kept constant by addition of appropriate
amounts of the parental empty expression vector (pRC/cmv). For nuclear
extracts, HepG2 cells were plated in 15-cm plates at a density of
8 × 106 cells/plate and serum-starved for 1-3 days;
and after two washings with cold phosphate-buffered saline, nuclear
extracts were prepared according to the method of Lee et al.
(30). Briefly, 1 ml of ice-cold extraction buffer (10 mM
Hepes (pH 7.9), 1.5 mM MgCl2, 10 mM
KCl, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml
leupeptin, and 1 µg/ml aprotinin) was added to the plates. After
three cycles of freeze-thawing, cytoplasmic extracts were recovered by
centrifugation at 12,000 rpm for 5 min, and pellets were resuspended in
buffer containing 20 mM Hepes (pH 7.9), 1.5 mM
MgCl2, 420 mM KCl, 0.2 mM EDTA,
25% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. Following a 30-min
incubation at 4 °C, nuclear extracts were spun down at 12,000 rpm
for 5 min.
Electrophoretic Mobility Shift Assays (EMSAs)--
Nuclear
extracts were preincubated for 5 min at room temperature in 25 mM NaCl, 10 mM Tris (pH 7.5), 1 mM
MgCl2, 5 mM EDTA (pH 8), 5% glycerol, and 1 mM dithiothreitol with 1 µg of poly(dI-dC) as a
nonspecific competitor. Where indicated, 5-µg extracts were preincubated for 1.5 h at 4 °C with 1 µg of polyclonal
antibodies (C20) directed against STAT3 in the presence of 1% Brij 96. A double-stranded nucleotide containing a STAT3 consensus binding site
derived from the c-fos gene (siem
(sis-inductible element), 5'-CATTTCCCGTAAATCTTGTCG-3') was
end-labeled using T4 kinase, and 10 pg of probe (20,000 cpm) was then
added to the protein mixture for 15 min. Samples were loaded on a 5%
polyacrylamide gel (30:1) and separated by electrophoresis in 50 mM Tris, 0.38 M glycine, and 1 mM
EDTA (pH 8.5). Gels were then dried and visualized by autoradiography.
Immunoprecipitation and Western Blot Analysis--
Nuclear cell
extracts (1-5 mg) were immunoprecipitated with the indicated
antibodies overnight at 4 °C on a rotator, followed by addition of
40 µl of protein A-Sepharose for 1 h at 4 °C. Cyclin D1 was
immunoprecipitated in the presence of 1% Brij 97, whereas STAT3
proteins were recovered in the presence of 1% Nonidet P-40. Immunoprecipitates were washed three times with lysis buffer
(0.1 M KHPO4 (pH 7.8) and 0.1% Triton
X-100), boiled for 5 min, and loaded on polyacrylamide gel. Following
electrotransfer, blots were washed five times and incubated overnight
at 4 °C in Tris-buffered saline (10 mM Tris (pH 8) and
150 mM NaCl) containing 6% bovine serum albumin. Blots
were then incubated with the indicated antibodies in Tris-buffered
saline containing 6% bovine serum albumin and 0.1% Tween, washed five
times, and further incubated with antibodies conjugated to horseradish
peroxidase. Proteins were visualized using the ECL system (Amersham
Pharmacia Biotech) according to the instructions of the manufacturer.
Fusion Protein Purification and Pull-down
Experiments--
Bacterial cultures (Escherichia coli
strain BL21 or DH5) expressing fusion proteins were grown in LB medium
and induced with 1 mM
isopropyl- Luciferase Assays--
HepG2 cells were plated in 24-well plates
24 h prior to transfection at a density of 3 × 104 cells/well. Transfected cells were washed twice with
ice-cold phosphate-buffered saline, and 150 µl of lysis buffer was
added to the wells. Extracts were then used directly to measure
luciferase activity by integrating total light emission over a 10-s
period using a Packard Topcount scintillation counter. Luciferase
activity was normalized based on protein concentrations.
In Vivo Interaction between Cyclin D1 and STAT3 Proteins--
To
determine whether cyclin D1 might be involved in the regulation of
STAT3 activation, we first investigated whether it could bind
specifically to STAT3 proteins. Following IL-6 stimulation, nuclear
extracts were recovered from HepG2 hepatoma cells, and co-immunoprecipitations were performed alternatively with polyclonal antibodies directed against cyclin D1 (Fig.
1A, lane 2),
polyclonal antibodies directed against STAT3 (lane 4), or
nonspecific antibodies (lanes 1 and 3). Proteins
present in the immunoprecipitates were revealed by immunoblotting with
the reciprocal antibodies. In both cases, cyclin D1 and STAT3 were
found to co-immunoprecipitate (Fig. 1A, compare lanes
1 and 3 with lanes 2 and 4).
Similar effects were also observed in a different cell line that
expresses the IL-6 receptor transducing complex, the human
erythroleukemia TF1 cell line (Fig. 1B). This
interaction was dependent on the presence of IL-6 since a very weak
interaction was detected between the two proteins when cells were
serum-starved for 2 days. By contrast, cyclin D1 and STAT3
co-immunoprecipitated in response to IL-6 stimulation (Fig.
1C, lanes 1-4). Whether the
phosphorylation has a direct effect on the interaction or is only
related to an increased quantity of STAT3 in the nucleus remains to be
determined. Importantly, these co-immunoprecipitations were carried out
using nuclear extracts from non-transfected cells; therefore, the
association between cyclin D1 and STAT3 does not require that these
proteins be overexpressed. Altogether, these results indicate that
STAT3 signaling proteins interact with cyclin D1.
Inhibition of STAT3 Transcriptional Activity by Cyclin
D1--
Recent results have shown that cyclin D1 can inhibit the
transcriptional activity of the MyoD, v-Myb, and DMP1 transcription factors (23-26). Having shown that cyclin D1 and STAT3 could interact, we therefore hypothesized that this interaction might affect the transcriptional activity of STAT3. To verify this, HepG2 cells were
cotransfected with a reporter construct containing two STAT3 consensus
binding sites upstream of a thymidine kinase minimal promoter together
with vectors expressing STAT3 and cyclin D1. Following transfection,
cells were serum-starved and stimulated with IL-6, and luciferase
activity was measured after 15 h in cytoplasmic extracts.
Inclusion of a STAT3 expression vector in the transfection mixture led
to a 6-fold increase in expression following cell stimulation (Fig.
2A, bars 1 and
2). Activation by STAT3 was inhibited in the presence of a
cyclin D1 expression vector (Fig. 2A, compare bars
2 and 3). Importantly, cyclin D1 was also able to block
transcriptional activation in the absence of a STAT3 expression vector,
suggesting that the endogenous protein is also inhibited (Fig.
2A, compare bars 4-6). The same effect was also
observed in the presence of leukemia inhibitory factor (Fig.
2B, compare bars 2 and 3) and
oncostatin M (Fig. 2C, compare bars 2 and
3), indicating that the cyclin D1 inhibitory effect is
shared by cytokines using the STAT3 signaling pathway. The cyclin
D1-mediated inhibitory effect on IL-6- and leukemia inhibitory factor-induced transactivation increased when increasing amounts of
cyclin D1 expression vectors were included in the transfection mixture
(Fig. 2D, compare bars 2-4 with bars
6-9). Thus, we concluded from these results that cyclin D1 can
inhibit the transcriptional activity of STAT3 proteins.
Effect of Cyclin D1 on STAT3 DNA Binding in COS Cells--
To
explain the effect of cyclin D1 on STAT3 transcriptional activity, we
first hypothesized that cyclin D1 might inhibit the DNA-binding
activity of the transcription factor. To obtain a high transfection
efficiency (>80%), COS-7 cells were transfected with vectors
expressing the IL-6 receptor together with plasmids expressing STAT3
and cyclin D1. Under these experimental conditions, STAT3 DNA binding
was induced following IL-6 stimulation (Fig. 3A, compare lanes 1 and 2). EMSA experiments were conducted in the presence of
antibodies against STAT3 to prove that the binding was specific for
this protein (Fig. 3A, compare lanes 4 and
5). Overexpression of cyclin D1 did not affect STAT3
DNA-binding activity (Fig. 3A, compare lanes 2 and 3), although cyclin D1 was clearly overexpressed
(compare lanes 7 and 8 in the lower
panel) and did interact with STAT3 (compare lanes 7 and
8 in the upper panel). In line with these
results, Western blot experiments indicated that overexpression of
cyclin D1 in COS cells did not affect the nuclear expression of STAT3
or its phosphorylation on Tyr705 (Fig. 3B,
lanes 2, 3, 5, and 6).
Thus, these results might indicate that cyclin D1 has no effect on
STAT3 DNA binding in COS cells. However, this conclusion depends on the
stoichiometry, and it remains to be determined if all STAT3 proteins
are in complex with cyclin D and whether STAT3 is capable of binding
both cyclin D1 and DNA simultaneously.
The Inhibitory Effect of Cyclin D1 Is Mediated through Its
Interaction with the Activation Domain of STAT3--
Following the
study of the effect of cyclin D1 on STAT3 DNA binding, we then
hypothesized that it should inhibit the activity of a chimeric
Gal4-STAT3 fusion protein. Using a Gal4-dependent luciferase reporter gene, we found that cyclin D1 was able to repress
transactivation by Gal4-STAT3 (Fig. 4,
compare bars 2 and 3). Importantly, cyclin D1 had
no effect on the transcriptional activity of a control Gal4-VP16 fusion
protein (Fig. 4, compare bars 5 and 6). This
result further suggested that the effect of cyclin D1 was probably not
mediated through inhibition of DNA binding and also indicated that
cyclin D1 may interact with the carboxyl-terminal activation domain of
STAT3. To verify this, in vitro pull-down experiments were
performed using bacterially produced full-length GST-cyclin D1 protein
and His6-tagged STAT3 containing amino acids 716-770
corresponding to the activation domain of STAT3 (STAT3
Thus, we concluded from these results that cyclin D1 binds at least to
the carboxyl-terminal activation domain of STAT3. Moreover, these
pull-down experiments also suggest that the interaction between cyclin
D1 and STAT3 is probably direct, although we cannot rule out the
possibility that cyclin D1 functions via another partner that could be
copurified from the bacteria with His-tagged STAT3 or GST-cyclin D1.
Cyclin D1 Inhibits STAT3-mediated Transactivation Independently of
Its Cdk4 Partner--
We then verified the specificity of the cyclin
D1 inhibitory effects among the other cyclins. STAT3 activity is
down-regulated in HepG2 cells during the first 6 h of IL-6
stimulation (31). To ascertain the physiological significance of the
results, we reasoned that any cyclin that would affect the activity of
STAT3 proteins should be expressed when the transcription factor is present in the nucleus. Among the different cyclins, cyclin A is
induced at the beginning of the S phase of the cell cycle, whereas
cyclin B appears at the end of the S phase and in G2. Therefore, these proteins are unlikely to modulate STAT3 activity. By
contrast, cyclin D1 is induced in early G1, and cyclin E
could be expressed in the middle of G1 (12). Therefore,
cyclin E could theoretically also affect the transcriptional activity
of STAT3. To verify this, HepG2 cells were cotransfected with a
luciferase reporter construct together with plasmids expressing STAT3
and cyclin D1 or cyclin E. Transcriptional activation by STAT3 was almost completely abolished in the presence of a cyclin D1 expression vector, whereas cyclin E was unable to affect STAT3 proteins, confirming the specificity of cyclin D1 inhibition (Fig.
6A, compare bars
2-4).
To extend these results, we then investigated if Cdk4, the kinase
partner of cyclin D1, was required in this inhibitory effect. To this
end, we used a mutant of cyclin D1 (cyclin D1-KE) that carries a
mutation in the cyclin box changing the lysine at amino acid 114 to
glutamic acid. This alteration resulted in a mutant cyclin D1-KE
protein that fails to bind to Cdk4 (32). Like wild-type cyclin D1, the
cyclin D1-KE mutant inhibited transcriptional activation by STAT3 (Fig.
6B, bar 4). Importantly, EMSA experiments
indicated that overexpression of cyclin D1-KE might not affect STAT3
DNA-binding activity in COS cells, confirming the results obtained with
wild-type cyclin D1 (Fig. 6B, compare lanes 5 and
7). As previously reported for the v-Myb and DMP1
transcription factors (24, 25), the inhibition observed with the cyclin
D1-KE mutant was consistently greater than that with the wild-type
cyclin D1 (Fig. 6B, compare bars 3 and
4). It is conceivable that competition for cyclin D1 binding
by endogenous Cdk4 might limit the inhibitory effect of cyclin D1 and
its interaction with STAT3. If so, this would explain why the cyclin
D1-KE mutant is a more potent STAT3 inhibitor that wild-type cyclin D1.
To confirm these results, we also used a specific Cdk4 inhibitor,
p16INK4. INK4 can compete with cyclin D1 for binding to Cdk4, but can
also inhibit Cdk4 without dissociating cyclin D1, resulting in
INK4·cyclin D·Cdk4 complexes (33). Significantly, cotransfection of
p16INK4 had no effect on the cyclin D1-mediated inhibition of STAT3
transcriptional activity (Fig. 6C, compare bars 3 and 4), further suggesting that the inhibitory effect is mediated by "free" cyclin D1. Taken together, these results suggest that cyclin D1-mediated inhibition of the transcriptional activity of
STAT3 is independent of the ability of cyclin D1 to activate Cdk4.
Cyclin D1 Reduces STAT3 Nuclear Levels--
As a first attempt to
characterize the inhibitory effect of cyclin D1 on STAT3
transcriptional activity in HepG2 cells, we hypothesized that cyclin D1
may also regulate the nuclear level of STAT3 in this cell line. To test
this hypothesis, HepG2 cells were stably transfected with an expression
vector encoding cyclin D1 or the corresponding parental plasmid.
Following G418 selection, cyclin D1 was clearly overexpressed compared
with control clones (Fig. 7A,
lanes 1 and 2). STAT3 proteins, once activated in
response to IL-6, become phosphorylated on Tyr705,
dimerize, and translocate to the nucleus. To verify if the
down-regulation of STAT3 transcriptional activity could be related to
reduced nuclear expression, Western blot experiments were performed on cytoplasmic and nuclear extracts. Following IL-6 stimulation, cytoplasmic STAT3 was expressed (Fig. 7B, upper
panel) and phosphorylated on Tyr705 in control cells
as well as in two different clones overexpressing cyclin D1 (Fig.
7C, compare lanes 1-3 with lanes
4-9). However, the cytoplasmic phosphorylation of STAT3 on
Tyr705 was reduced in clone 1 (Fig. 7C, compare
lanes 2 and 3 with lanes 5 and
6). In the presence of cyclin D1, the expression level of STAT3 in the nucleus was very weak (Fig. 7B, lower
panels, compare lanes 1-3 with lanes 4-9),
but remained normal under control conditions. Interestingly, STAT3 was
almost absent from the nucleus of clone 2, and these cells were found
to express higher levels of cyclin D1 (data not shown). Importantly,
this effect was not due to a general defect of STAT3 translocation
since nuclear expression of the transcription factor could be detected
upon a longer exposure of the blot (Fig. 7D, compare
lanes 1-4 with lane 5).
We concluded from these results that cyclin D1 decreased the
steady-state level of STAT3 proteins in the nucleus. Thus, this down-regulation might explain the negative effect of cyclin D1 on STAT3
transcriptional activity in HepG2 cells. If this hypothesis is correct,
we then reasoned that cyclin D1 should not interact with STAT3 at the
time of its maximal activation, whereas STAT3 should disappear from the
nucleus with the appearance of cyclin D1·STAT3 complexes. To verify
this, HepG2 cells were stimulated with IL-6, and nuclear activation of
STAT3 was evaluated in parallel with the appearance of cyclin
D1·STAT3 complexes. After 15 min, STAT3 translocated to the nucleus
and was phosphorylated on Tyr705 and Ser727;
however, no interaction could be detected between STAT3 and cyclin D1
in co-immunoprecipitation experiments (Fig. 7E, lanes 1 and 2). Interestingly, cyclin D1 and STAT3 were found
to interact after 2 h of stimulation, when the nuclear level of
STAT3 and its phosphorylation status were found to decrease (Fig.
7E, lanes 2 and 3). These results,
together with the evidence regarding the interaction of STAT3 and
cyclin D1, led us to propose that the transcriptional inhibition of
STAT3 by cyclin D1 could be mediated through reduced nuclear expression
in HepG2 cells.
The results presented in this study describe a new pathway for
inhibiting STAT3 activation and establish a new role for cyclin D1 as a
STAT3 inhibitor. We have shown that cyclin D1 can interact with STAT3
proteins and block their activity, suggesting a novel biological role
for cyclin D1 in the feedback regulation of IL-6-type cytokine
signaling pathways.
At the nuclear level, STAT3 activation is transient, and activated
transcription factors disappear from the nucleus within 1-6 h after
ligand stimulation (34). Removal of activated STAT proteins from the
nucleus has been shown to require phosphatase action, with reappearance
of the STAT in the cytoplasm in a dephosphorylated state (35).
Phosphorylated STAT proteins can also be detected in association with
ubiquitin, and proteasome inhibitors prolong the activation of these
transcription factors, suggesting that proteolytic degradation
participates in the negative regulation of nuclear STAT activation
(36). Association with inhibitory molecules has also been recently
described: the PIAS family proteins are negative regulators of STAT
activity, and they associate with STAT proteins and inhibit DNA binding
(37). Additional stimuli can also block the STAT signaling cascade in
the nucleus, and we have recently shown that the cell cycle inhibitor
p21WAF1/CIP1/SDI1 also blocks the IL-6 signaling pathway
through a feedback mechanism (31). Thus, multiple levels of regulation
act upstream or at the level of STAT3 proteins, and the results
presented in this study extend the observation that gene activation by
STAT3 proteins is affected by signals that control cell cycle progression.
Cell cycle progression is activated by the binding of D-type cyclins to
Cdk4 and Cdk6; therefore, cyclin D1 is generally considered a Cdk
activator (13). However, the cyclin D1-mediated repression of STAT3
transcriptional activity is unrelated to its function as a Cdk4
regulatory subunit, whereas cyclin E is unable to affect STAT3
proteins. Therefore, the transcriptional activity of cyclin D1 is
probably unique among the cyclins and is not mediated by Cdk4.
Importantly, similar observations have been reported for the estrogen-,
v-Myb-, DMP1-, and MyoD-regulated genes, where cyclin D1 regulates the
transcriptional activity independently of Cdk4 (23-26). Taken
together, these observations indicate that cyclin D1 can act via two
different mechanisms: as a Cdk activator, it regulates cell cycle
progression; and as a transcriptional regulator, it modulates the
activity of transcription factors (19). This effect remains to be
clarified, however, since cyclin D1 is a positive regulator of estrogen
receptors, whereas it inhibits the activity of the v-Myb, DMP1, MyoD,
and STAT3 transcription factors.
A few possibilities can be raised concerning the molecular mechanisms
whereby STAT3 is inhibited by cyclin D1. The cyclin is able to inhibit
the transcriptional activity of a chimeric Gal4-STAT3 protein
containing the activation domain of STAT3. The domain that modulates
the interaction between cyclin D1 and STAT3 proteins remains to be
fully identified; but, as a first attempt to map this region, we have
shown that cyclin D1 is at least able to interact with the activation
domain of STAT3. In light of this result, we might speculate that
cyclin D1 could occlude this domain, thereby blocking the interactions
of STAT3 with the RNA polymerase II transcriptional machinery or with
its essential cofactors such as CBP/p300 (38). Steric hindrance would,
in this case, lead to transcriptional repression. Interestingly, CBP/p300 has been recently shown to bind the carboxyl-terminal part of
STAT3 (38), and cyclin D1 has been shown to interact with essential
transcriptional coactivators (22). Additionally, the binding of cyclin
D1 within the activation domain of STAT3 could inhibit the
phosphorylation of serine 727, a critical residue involved in the
transcriptional activity of STAT3 (39). Again, interaction of cyclin D1
with STAT3 could reduce the accessibility to Ser727.
A second hypothesis can probably be raised concerning the molecular
mechanisms whereby STAT3 signaling is inhibited, as we found that
cyclin D1 decreased the steady-state level of STAT3 proteins in the
nuclei of HepG2 cells. This was not a general defect since STAT3 can
enter the nucleus in the presence of cyclin D1, but to a lesser extent.
These observations raise the possibility that STAT3 nuclear
localization is regulated following its association with cyclin D1,
maybe in a cell-cycle dependent manner. In line with this hypothesis,
the transcriptional activity of STAT1 has also been shown to be
differently regulated during cell cycle progression (40). This effect
remains to be clarified, but we speculate that STAT3 inhibition is
mediated through two potential mechanisms: occlusion of the
transactivation function and a decrease in nuclear amount.
Altogether, these results point to a novel biological role for cyclin
D1 as an important inhibitor of STAT3 transcriptional activity. We
propose a model by which IL-6 stimulation induces the interaction
between cyclin D1 and STAT3 proteins, leading to the feedback
down-regulation of STAT3 activation. These findings extend the role of
cyclin D1 as a converging point of several intracellular signaling cascades.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside after the absorbance had reached 0.6. Following centrifugation, bacterial pellets were lysed
by sonication in phosphate-buffered saline containing 1% Triton X-100
and 1 mM EDTA. Extracts were recovered by centrifugation at
12,000 rpm for 5 min, and fusion proteins were purified by affinity
chromatography following the procedures provided by the suppliers.
100-200 ng of purified His-STAT3
1-716 (see Fig. 5) was mixed with
100-200 ng of GST or GST-cyclin D1 coupled to glutathione beads and
incubated for 30 min at 4 °C in binding buffer (20 mM Tris-HCl (pH 7.5), 137 mM NaCl. Beads were then washed
three times with binding buffer and one time with 50 mM
Tris-HCl (pH 8), boiled for 5 min, and loaded on polyacrylamide gel for
Western blot analysis. The C20 polyclonal antibody was used to detect
His-STAT3
1-716, corresponding to the carboxyl-terminal part of STAT3.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
In vivo association between cyclin
D1 and STAT3 proteins. A, HepG2 cells were
serum-starved for 48 h and then stimulated with IL-6 (10 ng/ml)
for 2 h. Nuclear cell extracts were immunoprecipitated
(IP) with polyclonal antibodies directed against cyclin D1
proteins (lane 2) or a control serum (lane 1),
separated by SDS-polyacrylamide gel electrophoresis, transferred to a
nitrocellulose filter, and probed with polyclonal antibodies directed
against STAT3 proteins (lanes 1 and 2).
Reciprocal immunoprecipitations were performed with polyclonal
antibodies directed against STAT3 proteins (lane 4) or a
control serum (lane 3), followed by membrane blotting with
polyclonal antibodies directed against cyclin D1 proteins (lanes
3 and 4). B, TF1 cells were serum-starved
for 24 h and then stimulated with IL-6 (10 ng/ml) for 2 h.
Immunoprecipitations were performed as described for A. C, HepG2 (lanes 1 and 2) or TF1 cells
(lanes 3 and 4) serum-starved for 24-48 h were
either stimulated for 2 h with IL-6 (10 ng/ml) (lanes 2 and 4) or left untreated (lanes 1 and
3). Nuclear extracts were then immunoprecipitated with
polyclonal antibodies directed against cyclin D1 and probed with
polyclonal antibodies directed against the
Tyr705-phosphorylated form of STAT3 proteins. Note that TF1
cells died very rapidly upon serum starvation, so we were unable to
completely inactivate STAT3.
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Fig. 2.
Cyclin D1 inhibits the transcriptional
activity of STAT3. A, HepG2 cells were cotransfected
with a vector expressing a luciferase reporter gene containing two
copies of a STAT3 consensus binding site linked to a minimal thymidine
kinase (Tk) promoter in the presence (bars 3 and
6) or absence (bars 1, 2,
4, and 5) of a vector encoding for cyclin D1
(CD). A STAT3 expression vector was also included in the
reaction mixture in bars 1-3. Following transfection, cells
were serum-starved for 15 h and stimulated overnight with IL-6 (10 ng/ml) (bars 2, 3, 5, and
6). Cytoplasmic extracts were then prepared and processed to
measure luciferase activity. The mean of nine transfections is shown.
B and C, HepG2 cells were transfected as
described for A, and cells were serum-starved for 15 h
and stimulated overnight with leukemia inhibitory factor
(LIF; 10 ng/ml) (B) or oncostatin M
(OSM; 10 ng/ml) (C). Cytoplasmic extracts were
then prepared and processed to measure luciferase activity.
D, HepG2 cells were transfected with different
concentrations of a cyclin D1 expression vector as described for
A, and cells were serum-starved for 15 h and stimulated
overnight with IL-6 or leukemia inhibitory factor (10 ng/ml).
Cytoplasmic extracts were then prepared and processed to measure
luciferase activity.
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Fig. 3.
Effect of cyclin D1 on STAT3 DNA-binding
activity. A, COS cells transfected with vectors
expressing the IL-6 receptor, gp130, and STAT3 were cotransfected with
empty parental vector (lanes 2 and 4-7) or
vector expressing cyclin D1 (lanes 3 and 8).
Following transfection, cells were serum-starved for 24 h and
stimulated for 15 min (IL-6 (10 ng/ml) plus IL-6 soluble receptor (1 µg/ml)) (lanes 2-5, 7, and 8).
Nuclear extracts were then prepared and analyzed by EMSAs in the
presence or absence of polyclonal antibodies directed against STAT3
proteins (lanes 4 and 5). In parallel, nuclear
extracts (1 mg) were immunoprecipitated (IP) with polyclonal
antibodies directed against cyclin D1 proteins, separated by
SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose
filter, and probed with polyclonal antibodies directed against STAT3
proteins ( lanes 6-8 in the upper panel), or
polyclonal antibodies directed against cyclin D1 proteins (lanes
6-8 in the lower panel). B, COS cells were
cotransfected as described for A, and nuclear extracts were
analyzed by Western blotting using polyclonal antibodies against STAT3
(lanes 1-3) or STAT3 phosphorylated on Tyr705
(lanes 4-6).
1-716) (Fig.
5A). We found that His-tagged STAT3
1-716 could be retained by a GST-cyclin D1 fusion protein immobilized on glutathione beads, whereas it did not bind to GST beads
alone (Fig. 5B, compare lanes 1 and
2). To confirm this result, we also determined that
GST-cyclin D1 could be retained by His-tagged STAT3
1-716
immobilized on beads, whereas it bound weakly to histidine beads alone
(Fig. 5B, compare lanes 3 and 4).
Pull-down experiments were also performed using HepG2 nuclear extracts
mixed with immobilized GST or GST-cyclin D1. The results indicated that
the endogenous STAT3 protein could also interact with GST-cyclin D1
(Fig. 5B, compare lanes 5 and 6).
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Fig. 4.
Cyclin D1 inhibits the transcriptional
activity of a Gal4-STAT3 fusion protein. HepG2 cells were
cotransfected with vectors expressing a Gal4-luciferase reporter gene
(500 ng) together with vectors expressing a Gal4 fusion protein linked
to the STAT3 activation domain (500 ng) in the presence (bar
3) or absence (bars 1 and 2) of a plasmid
encoding cyclin D1 (CD; 10 ng). The same experiments were
performed in parallel using a Gal4-VP16 plasmid (500 ng) (bars
4-6). Following transfection, cells were serum-starved for
15 h and stimulated overnight with IL-6 (10 ng/ml). Cytoplasmic
extracts were then prepared and processed to measure luciferase
activity. The mean of four transfections is shown.
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Fig. 5.
Cyclin D1 interacts with the activation
domain of STAT3. A, shown is a representation of the
carboxyl-terminal STAT3 1-716 fusion protein used in the
pull-down experiments. B, His-tagged STAT3 fusion proteins
corresponding to the activation domain of STAT3 (STAT3
1-716;
100-200 ng) were tested for binding to GST or to full-length
GST-cyclin D1 (100-200 ng) immobilized on Sepharose beads (lanes
1 and 2). Samples were then washed four times and
separated on 6% polyacrylamide gels, and STAT3 binding was detected by
Western blotting using anti-STAT3 polyclonal antibodies (C20). Purified
GST-cyclin D1 proteins (20 ng) were incubated for 30 min at 4 °C
with histidine or His-tagged STAT3 immobilized on nickel-agarose beads
(20 ng) (lane 3 and 4). Samples were then washed
four times and separated on 6% polyacrylamide gels, and cyclin D1
binding was detected by Western blotting using anti-cyclin D1
polyclonal antibodies. In parallel, HepG2 cells were serum-starved for
48 h and then stimulated with IL-6 (10 ng/ml) for 2 h.
Nuclear cell extracts (10-50 µg) were incubated for 30 min at
4 °C with GST or GST-cyclin D1 immobilized on Sepharose beads
(lanes 5 and 6). STAT3 binding was detected by
Western blotting using anti-STAT3 polyclonal antibodies.
View larger version (17K):
[in a new window]
Fig. 6.
Specific action of cyclin D1 on STAT3
transcriptional activity. A, HepG2 cells were
cotransfected as described in the legend to Fig. 2 together with
vectors expressing STAT3 alone (bars 1 and 2) or
with cyclin D1 (CD; bar 3) or cyclin E
(CE; bar 4). Following transfection, cells were
serum-starved for 15 h and stimulated overnight with IL-6 (10 ng/ml) (bars 2-4). Cytoplasmic extracts were then prepared
and processed to measure luciferase activity. The mean of nine
transfections is shown. B, HepG2 cells were cotransfected as
described for A with vectors expressing STAT3 alone
(bars 1 and 2) or with cyclin D1 (bar
3) or a mutant of cyclin D1 unable to bind Cdk4 (cyclin D1-KE
(CD-KE)) (bar 4). Luciferase activity was
evaluated as described for A. The mean of eight
transfections is shown. In parallel, COS cells transfected with vector
DNA expressing the IL-6 receptor were transfected with STAT3 vectors in
the presence (lane 5) or absence (lanes 6 and
7) of a vector expressing cyclin D1-KE. Following
transfection, cells were serum-starved for 24 h and stimulated as
described in the legend to Fig. 3 (lanes 5 and 7)
or left untreated (lane 6). Nuclear extracts were then
prepared and analyzed by EMSAs using radiolabeled oligonucleotides
encoding a STAT3 consensus binding site. C, HepG2 cells were
cotransfected as described for A with vectors expressing
STAT3 alone (bars 1 and 2) or with cyclin D1
(bar 3) or cyclin D1 and the p16INK4 inhibitor
(p16) (bar 4). Luciferase activity was evaluated
as described for A. The mean of eight transfections is
shown. Tk, thymidine kinase.
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[in a new window]
Fig. 7.
Cyclin D1 reduces the STAT3 nuclear
level. A, HepG2 cells were stably transfected with an
expression vector encoding cyclin D1 (lane 2) or the
corresponding parental plasmid (lane 1). Following G418
selection, overexpression was verified using nuclear extracts analyzed
by Western blotting and polyclonal antibodies against cyclin D1.
B, HepG2 cells described for A were serum-starved
for 48 h and then stimulated with IL-6 (10 ng/ml) (lanes
2 and 3) for the indicated times (minutes
(mn)). Nuclear (lower panel) and cytoplasmic
(upper panel) extracts were then prepared, and the
expression of STAT3 was analyzed by Western blotting in control clones
(lanes 1-3) or in cyclin D1-overexpressing cells
(lanes 4-6 (clone 1) and 7-9 (clone 2)).
C, stable cell lines were stimulated as described for
B, and cytoplasmic activation of STAT3 was analyzed by
Western blotting using antibodies directed against its
Tyr705-phosphorylated form. D, HepG2 cells
overexpressing cyclin D1 (lanes 1-4) and a control clone
(lane 5) were serum-starved for 48 h and then
stimulated with IL-6 (10 ng/ml) (lanes 2-5) for the
indicated times. Nuclear expression of STAT3 was analyzed by Western
blotting, and two blot exposition times are presented. E,
HepG2 cells were serum-starved for 48 h and then stimulated with
IL-6 (10 ng/ml) (lanes 2 and 3) for the indicated
times. Nuclear cell extracts were immunoprecipitated (IP)
with polyclonal antibodies directed against cyclin D1 proteins,
separated by SDS-polyacrylamide gel electrophoresis, transferred to a
nitrocellulose filter, and probed with polyclonal antibodies directed
against STAT3 Tyr705 (fourth panel). In
parallel, nuclear activation of STAT3 was analyzed using antibodies
directed against STAT3 (first panel) or its
Ser727 (third panel)- or Tyr705
(second panel)-phosphorylated forms.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Drs. R. Bernards, F. Gouilleux, D. E. Levy, and P. Whyte for the gift of various expression vectors and Dr. A. Lundquist for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by a fellowship (to F. B.) and a grant from the Ligue Nationale pour la Recherche sur le Cancer ("Equipe Labelisée la Ligue contre le Cancer").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.
To whom correspondence should be addressed. Tel.:
33-2-41-35-47-33; Fax: 33-2-41-73-16-30; E-mail:
olivier.coqueret@univ-angers.fr.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M100795200
2 Details of constructs are available on request.
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ABBREVIATIONS |
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The abbreviations used are: JAK, Janus kinase; STAT, signal transducers and activators of transcription; IL-6 interleukin-6, Cdk, cyclin-dependent kinase; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay; CBP, cAMP response element-binding protein-binding protein.
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