From the Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G12 8QQ, United Kingdom
Received for publication, June 21, 2000, and in revised form, October 4, 2000
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ABSTRACT |
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Increased rates of RNA polymerase (pol) III
transcription constitute a central feature of the mitogenic response,
but little is known about the mechanism(s) responsible. We demonstrate
that the retinoblastoma protein RB plays a major role in suppressing pol III transcription in growth-arrested fibroblasts. RB knockout cells
are compromised in their ability to down-regulate pol III following
serum withdrawal. RB binds and represses the pol III-specific transcription factor TFIIIB during G0 and early
G1, but this interaction decreases as cells approach S
phase. Full induction of pol III coincides with mid- to late
G1 phase, when RB becomes phosphorylated by cyclin D- and
E-dependent kinases. TFIIIB only associates with the
underphosphorylated form of RB, and overexpression of cyclins D and E
stimulates pol III transcription in vivo. The RB-related protein p130 also contributes to the repression of TFIIIB in
growth-arrested fibroblasts. These observations provide insight into
the mechanisms responsible for controlling pol III transcription during
the switch between growth and quiescence.
The retinoblastoma protein RB is a highly abundant tumor
suppressor that can bind and regulate a variety of transcription factors (reviewed in Refs. 1-4). One example that has been added recently to the growing list of RB-binding proteins is the RNA polymerase (pol)1
III-specific factor TFIIIB (5, 6). Recombinant RB was shown to bind to
TFIIIB in vitro and repress its activity (5, 6). Furthermore, coimmunoprecipitation and cofractionation experiments demonstrated a stable association between endogenous cellular RB and
TFIIIB (6). The functional significance of this interaction was shown
in studies of RB knockout mice, since primary fibroblasts from
Rb TFIIIB is required for the expression of all pol III templates
(reviewed in Refs. 8 and 9). It serves to recruit the polymerase to a
promoter and position it over the transcription start site (10). By
interacting with this general factor, RB appears able to regulate the
expression of all pol III-transcribed genes, including tRNA, 5 S rRNA,
U6 small nuclear RNA, VA1, and Alu genes (5, 6, 11). Since a high rate
of tRNA and rRNA synthesis is required to sustain rapid growth, it has
been speculated that the inhibition of pol III transcription may
contribute to the growth suppression capacity of RB (12-14).
RB function is regulated by cyclin-dependent kinases
(reviewed in Refs. 2 and 3 and Ref. 15). The cyclin
D-dependent kinases CDK4 and CDK6 phosphorylate RB
partially and the process is completed by cyclin E-CDK2 (16, 17). The
action of cyclin E-CDK2 appears to depend on prior phosphorylation by
the cyclin D-dependent kinases (17). At least 10 serine and
threonine residues can become phosphorylated in RB (2, 16). Once
hyperphosphorylated, RB loses its ability to bind to many of its
targets and function as a growth suppressor (2, 15). This occurs at the
G1/S phase transition, in parallel with the synthesis of
cyclins D and E (2, 15). The cyclin D-dependent kinases
become active in mid- to late G1, at a stage called the
restriction, or R, point when cells lose their serum dependence (18).
Cyclin E-CDK2 is activated shortly afterward, as cells leave
G1 phase (18). RB is then maintained in the
hyperphosphorylated state throughout S, G2, and M phases,
until it is dephosphorylated by protein phosphatase 1 at the end of
mitosis (15). In cycling cells, therefore, the underphosphorylated form
of RB is only present during the early period of G1.
However, it is also found in resting G0 cells, which do not
express significant levels of cyclins D and E (3).
The level of pol III transcription decreases significantly when growing
fibroblasts are deprived of serum (19, 20). This is likely to reflect a
diminished requirement for protein production. Although the switch
between G0 and G1 phases is the principle determinant of proliferation rate in mammalian cells, the molecular mechanism(s) responsible for regulating pol III activity during this
transition are largely uncharacterized. This constitutes an important
gap in our current understanding, since the rate of pol III
transcription will undoubtedly have a major influence on the growth and
proliferation of cells. One study concluded that a specific reduction
in TFIIIB activity was responsible for down-regulating pol III
transcription in growth-arrested cells, although the molecular details
were not determined (21). In contrast, another laboratory demonstrated
that HeLa cells down-regulate pol III transcription when grown in low
serum due to a decrease in the activity of TFIIIC2 (22, 23). This is
associated with a specific reduction in the levels of an essential
subunit called TFIIIC Cell Culture--
Balb/c 3T3 (A31), SV3T3 (Cl38), and mouse
embryonic fibroblast cells were all grown in Dulbecco's
modified Eagle's medium (DMEM; Life Technologies, Inc.) supplemented
with 10% fetal calf serum (FCS), 100 units/ml penicillin, and 100 µg/ml streptomycin and were harvested when subconfluent. An
insulin-transferrin-selenium supplement (Life Technologies) was added
to the medium used to grow mouse embryonic fibroblasts. Unless
otherwise specified, cell growth was arrested by reducing the serum
concentration to 0.5%; mitogenic stimulation was then induced with
20% serum.
Flow Cytometry--
Cells to be analyzed by flow cytometry were
harvested in dissociation buffer (Sigma) and fixed in
phosphate-buffered saline/ethanol (1:1, v/v). Propidium iodide (40 µg/ml) was added, and the DNA content of cell samples was measured
using a Becton Dickinson FACScan (10,000 events/sample). Data were
analyzed using Cell Quest software.
Thymidine Incorporation--
[3H]Thymidine (0.1 µCi/ml) was added to serum-stimulated or quiescent cells 3 h
prior to harvesting. Cells were then washed twice in phosphate-buffered
saline, three times in 5% trichloroacetic acid, and twice in ethanol.
Samples were solubilized in 0.3 M NaOH, and the
incorporation of [3H]thymidine into DNA was then measured
by liquid scintillation counting.
Phosphate Labeling in Vivo--
Subconfluent Balb/c 3T3 cells
were cultured for 24 h in DMEM containing 0.5% FCS. They were
then incubated for 15 h in phosphate-free DMEM containing 100 µCi/ml [32P]orthophosphate either in the absence of
FCS, to give G0 phase cells, or the presence of 10% FCS,
to give S phase cells; early G1 phase cells were generated
by adding 10% FCS for the final 3 h of the incubation. Cells were
then lysed in RIPA buffer (64 mM Hepes, pH 7.4, 150 mM NaCl, 50 mM NaF, 10 mM EDTA,
1.2% Triton X-100, 0.64% sodium deoxycholate, 0.128% SDS, 10 mM Northern Blotting and Nuclear Run-on--
Total cellular RNA was
extracted using TRI reagent (Sigma), according to the manufacturer's
instructions. Agarose gel electrophoresis, Northern transfer, and
hybridization were carried out as previously (24). The B2 gene probe
was a 240-base pair EcoRI-PstI fragment from
pTB14 (25). The tRNALeu gene was a 240-base pair
EcoRI-HindIII fragment from pLeu (25). The
acidic ribosomal phosphoprotein P0 (ARPP P0) probe was a 1-kilobase pair EcoRI-HindIII fragment from the mouse
cDNA (26). Nuclear run-on assays were carried out as previously
(11).
Transient Transfection--
Transient transfections used the
calcium phosphate precipitation method. DNA precipitates were left on
the plates overnight, and then the cells were washed with
phosphate-buffered saline and cultured for 24 h before harvesting.
Total RNA was extracted using TRI reagent (Sigma), according to the
manufacturer's instructions. It was then analyzed by primer extension
using primers for VA1 (5'-CACGCGGGCGGTAACCGCATG-3') and CAT
(5'-CGATGCCATTGGGATATATCA-3'), as described previously (11).
Plasmids--
The pVA1 plasmid contains the adenovirus VA1 gene
(27). pHu5S3.1, pLeu, and pU6/Hae/RA.2 contain human 5 S rRNA,
tRNALeu, and U6 gene promoters, respectively (25, 31).
Expression vectors Rc-CDK2, Rc-CDK4, Rc-cycD1, and Rc-cycE contain
CDK2, CDK4, cyclin D1, and cyclin E cDNAs, respectively, cloned
into the pRc-CMV vector (Invitrogen) downstream of the CMV immediate early promoter (28). pCMVp16 contains the p16 cDNA fused to a CMV
polyadenylation signal and cloned downstream of the CMV immediate early
promoter (29). Rz 89-12 contains a ribozyme against murine p16 mRNA
subcloned into the pX expression vector (30). pCAT (Promega) contains
the CAT gene driven by the SV40 promoter and enhancer.
Extracts, Protein Fractions, and Transcription
Assays--
Whole-cell extracts were prepared using a freeze-thaw
procedure described previously (31). HeLa nuclear extracts were
purchased from the Computer Cell Culture Center (Mons). PC-B and PC-C
phosphocellulose step fractions were prepared as previously (31).
A25(0.15) fraction containing TFIIIB was prepared by chromatography on
phosphocellulose and DEAE-Sephadex, as previously (31). CHep-1.0
fraction containing TFIIIC and pol III was prepared by sequential
chromatography on phosphocellulose and heparin-Sepharose, as previously
(31). Recombinant TBP was purchased from Promega.
Transcription reactions were carried out as previously (25), except
that pBR322 was not included, and the incubations were for 60 min at
30 °C.
Immunoprecipitation--
Whole cell extract (150 µg) was
incubated for 4 h at 4 °C on an orbital shaker with 20 µl of
protein A-Sepharose beads carrying equivalent amounts of prebound IgG.
Samples were then pelleted, supernatants were removed, and the beads
were washed five times with 150 µl of LDB buffer (25). The bound
material was analyzed by Western blotting. In the experiment shown in
Fig. 7A, reticulocyte lysate (15 µl) containing RB
translated in the presence of [35S]Met and
[35S]Cys was treated for 10 min at 30 °C with or
without a mixture of baculovirus-expressed cyclin D-CDK4, cyclin
E-CDK2, and cyclin A-CDK2; it was then incubated for 4 h with
whole cell extract (150 µg) during immunoprecipitation. In this case,
the precipitated material was analyzed by autoradiography rather than
Western blotting.
Antibodies and Western Blotting--
Antibodies used were C-15
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and G3-245
(Pharminogen) against RB, C-20 (Santa Cruz Biotechnology) against p130,
monoclonal antibody clone 46 (Transduction Laboratories) against
TFIIIC Reverse Transcriptase-PCR Analysis--
RNA was extracted using
TRI Reagent (Sigma), according to the manufacturer's specifications.
Reverse transcription reactions were performed for 1 h at 42 °C
using 3 µg of RNA, 200 ng of Random Hexamers (Promega), and 400 units
of Superscript II Reverse Transcriptase (Life Technologies) in a total
volume of 40 µl of 1× First Strand Buffer (Life Technologies)
containing 10 mM dithiothreitol and a 0.5 mM
concentration of each dNTP.
PCRs were carried out using a PTC-100 programmable thermal controller
(MJ Research Inc). 2 µl of cDNA was amplified with 20 pmol of
either TFIIIC
Reaction products were resolved on a 2% agarose gel and visualized by
ethidium bromide staining.
Pol III Transcription Decreases When Fibroblasts Are Deprived of
Serum--
Actively growing Balb/c 3T3 cells were made quiescent by
serum withdrawal. The majority of cells had arrested in a
G0/G1 phase state after 1 day of culture under
serum-free conditions, as indicated by flow cytometric analyses of
their DNA content (Fig. 1A,
0 h serum stimulation). This conclusion was supported by
measurements of thymidine incorporation into newly synthesized DNA
(Fig. 1B). The abundance of pol III transcripts derived from
the B2 middle repetitive gene family was substantially reduced in the
growth-arrested cells, as revealed by Northern blotting (Fig.
1C, upper panel, compare
lanes 9 and 10). This effect was
specific, since levels of a pol II transcript encoding ARPP P0 did not
diminish following serum deprivation (Fig. 1C,
lower panel).
The growth-arrested fibroblasts were stimulated to reenter the cell
cycle by the addition of medium containing 20% serum. Flow cytometric
analysis and thymidine incorporation measurements demonstrated that S
phase was reached between 12 and 15 h after serum stimulation
(Fig. 1, A and B). Northern blotting with a B2
gene probe revealed a slight increase in pol III transcript levels by
mid-G1 phase, 6-9 h after the addition of serum, and revealed that near maximal expression was reached by 12 h after mitogenic stimulation, shortly before S phase entry (Fig.
1C). We conclude that Balb/c 3T3 cells undergo growth arrest
within 24 h of serum withdrawal and that this is accompanied by a
significant reduction in pol III activity; when these fibroblasts
resume cycling, pol III activity is restored during late G1
phase. These observations are consistent with previous studies of 3T6
and BHK cells (19, 20, 35).
The Level of TFIIIC
Sinn et al. (23) reported previously that growth of HeLa
cells in 0.5% serum results in a specific decrease in the abundance of
the TFIIIB Activity Is Down-regulated and Limiting in Serum-starved 3T3
Cells--
Add-back experiments were carried out to determine which
factor is limiting for pol III transcription in extracts of 3T3 cells. A fraction containing partially purified TFIIIB was found to stimulate transcription when titrated into extracts of either growing or serum-starved cells (Fig. 3A).
This effect was highly specific, since little or no stimulation was
observed in response to a fraction containing TFIIIC and pol III. The
activity of all fractions was confirmed using complementation
assays.4 The data suggest
that under the conditions used, TFIIIB is limiting, while TFIIIC and
pol III are in relative excess. This implies that the rate of pol III
transcription in 3T3 cells may be dictated by the availability of
active TFIIIB. We therefore carried out complementation assays to
compare directly the activity of TFIIIB in extracts prepared from cells
harvested either before or after serum withdrawal. In these assays, the
extracts are subjected to mild heat treatment, which selectively
inactivates endogenous TFIIIC; they are then tested for their ability
to support transcription when mixed with a complementing system
containing excess TFIIIC, TBP, and pol III (31). Extracts of growing
3T3 cells were found to contain sufficient TFIIIB activity to allow
robust transcription in this assay. In contrast, equal amounts of
extract from serum-starved cells gave little or no expression above
background (Fig. 3B). We conclude that TFIIIB activity is
low and limiting in extracts of serum-deprived 3T3 cells.
Serum Deprivation Produces Little or No Change in the Levels of BRF
and TBP--
We investigated whether the abundance of TFIIIB changes
in response to serum. Western blotting showed that 72 h of serum
deprivation resulted in little or no change in the level of the BRF
subunit of TFIIIB (Fig. 4A).
Levels of the TFIIIB subunit TBP are also maintained after extended
periods without serum (Fig. 4B). As an internal control, we
also monitored the pol II-specific factor TFIIB in these extracts and
found that this too remained unchanged (Fig. 4C). Although
we cannot exclude the possibility that unidentified components of
TFIIIB become less abundant in growth-arrested fibroblasts, the
available evidence suggests that a decrease in the amount of TFIIIB is
not responsible for its loss of activity when Balb/c 3T3 cells are
cultured in the absence of serum.
RB Knockout Fibroblasts Are Compromised in Their Ability to
Down-regulate pol III Transcription following Exit from the Cell
Cycle--
Genetic experiments have demonstrated previously that RB
plays an important role in regulating pol III transcription in murine fibroblasts (11). We therefore addressed the possibility that RB may
contribute to the control of pol III during cell cycle withdrawal.
Fibroblasts derived from either wild-type or RB knockout mice were
maintained in 20% serum or made quiescent by transfer to 0.5% serum.
Previous studies have demonstrated that specific disruption of the
Rb gene does not prevent fibroblasts from withdrawing from
cycle following serum deprivation (26, 36). This was confirmed in the
current study by measuring the incorporation of thymidine into newly
synthesized DNA.2 Northern blot analysis revealed that tRNA
levels are elevated in the Rb
To reinforce the above data, we carried out nuclear run-on assays to
measure directly the effect of serum on rates of pol III transcription
in matched Rb+/+ and
Rb RB Binds to TFIIIB in Growth-arrested Fibroblasts, but This
Interaction Diminishes following Mitogenic Stimulation--
It has
been shown that TFIIIB is a specific target for repression by RB in
murine fibroblasts (6). Since the experiments above using knockout
cells provide evidence that RB contributes to the repression of pol III
following growth arrest, we carried out immunoprecipitation assays to
compare the level of interaction between RB and TFIIIB during
quiescence with that seen in fibroblasts that have been stimulated to
reenter the cell cycle. An antiserum against RB was used to
immunoprecipitate proteins from 3T3 cells that had been starved of
serum for 36 h or starved for 24 h and then refed with 20%
serum for various times prior to harvesting; the precipitates were then
probed by Western blotting for both RB (lower
panel) and the BRF subunit of TFIIIB (upper
panel). A substantial amount of BRF was found to
coprecipitate with RB from the quiescent cells (Fig.
6A). This coprecipitation
reflects a specific interaction with RB, since BRF was not detected in material immunoprecipitated with a control antiserum against the TAFI48 subunit of the pol I factor SL1. The association
between RB and BRF was maintained in cells harvested in early
G1 phase, 3 or 6 h after mitogenic stimulation.
However, the interaction had begun to diminish after 9 h and was
substantially reduced by late G1 phase, 12 h after the
serum addition. Fig. 6B shows quantitation of this and two
similar experiments, in which the amount of coprecipitated BRF at each
time point has been normalized to the total amount of RB in each
immunoprecipitation. It is apparent that the down-regulation of pol III
transcription that accompanies growth arrest correlates with an
interaction between TFIIIB and its repressor RB; when cells resume
cycling, this interaction is maintained during early G1
phase but decreases late in G1, in parallel with the
activation of pol III. We estimate that RB is bound to between 66 and
74% of TFIIIB present in growth-arrested fibroblasts but that binding
has diminished by 6-fold or more by 15 h after serum stimulation,
when most cells are in S phase.
TFIIIB Interacts Specifically with the Underphosphorylated Form of
RB--
Western blotting revealed no change in the abundance or
electrophoretic mobility of BRF that might account for its increased binding to RB in quiescent fibroblasts (Fig. 4A). In
contrast, RB becomes phosphorylated at multiple sites when resting
cells are stimulated to proliferate (16, 17, 37-39). More
specifically, RB is found in an underphosphorylated state during
G0 and early G1 phases but becomes heavily
phosphorylated by cyclin D- and cyclin E-dependent kinases
shortly before entry into S phase (reviewed in Refs. 2 and 3 and Ref.
15). Evidence of this can be seen in the lower
panel of Fig. 6A, where hyperphosphorylation leads to a decrease in the electrophoretic mobility of RB, beginning 9 h after mitogenic stimulation. The effect is seen more clearly in Fig. 6C, which shows a Western blot carried out using an
antibody that only recognizes RB when it is phosphorylated at serine
780. Since phosphorylation of RB correlates temporally with its
dissociation from TFIIIB, we looked for a causal link between these two phenomena.
Experiments were carried out to test whether pretreatment of RB with
cyclin-dependent kinases could influence its ability to
coimmunoprecipitate with TFIIIB. Radiolabeled RB was synthesized in vitro by translation with a reticulocyte lysate in the
presence of [35S]Met and [35S]Cys.
Pretreatment of this RB with a mixture of recombinant CDK2 and CDK4 and
their partner cyclins caused a decrease in its electrophoretic mobility, consistent with hyperphosphorylation (Fig.
7A, lanes 1 and 2). These forms of RB were incubated with a
cell-free extract, to allow interaction with the endogenous TFIIIB;
immunoprecipitations were then carried out using an anti-BRF antiserum.
Whereas the underphosphorylated RB was found to coimmunoprecipitate
with BRF, no interaction was observed with the RB that had been
preincubated with cyclin-dependent kinases (Fig.
7A, lanes 4 and 5). These data suggest that hyperphosphorylation of RB can prevent it from binding to TFIIIB.
To address this issue further, we examined the phosphorylation state of
the RB that is associated with TFIIIB. A population of RB molecules
copurifies extensively with TFIIIB because of the physical interaction
between these proteins (6). We compared the electrophoretic mobility of
the RB present in an unfractionated cell extract with that found
associated with partially purified TFIIIB (Fig. 7B). The RB
present in extracts of asynchronous cells migrates as a doublet in
SDS-polyacrylamide gels, because the hyperphosphorylated protein has a
retarded mobility compared with the underphosphorylated form (37-39).
In contrast, the RB present in the TFIIIB fraction runs as a single
tight band that comigrates with the higher mobility form detected in
crude extracts. This suggests that it is only the underphosphorylated
form of RB that copurifies with TFIIIB. To test this further, we used a
panel of antibodies that exclusively recognize RB that is
phosphorylated at particular sites (34). Serine 780 of RB is
phosphorylated in vivo specifically by the cyclin
D-dependent kinases (34, 40). An antibody that only reacts
with RB that is phosphorylated at serine 780 gives a strong signal when
used to probe crude extracts of asynchronous cells (Fig.
7C). In contrast, virtually no signal was obtained when this
antibody was tested in parallel with partially purified TFIIIB, despite
the fact that this same TFIIIB fraction contains readily detectable
amounts of RB, as shown in Fig. 7B. We conclude that the RB
which copurifies with TFIIIB is not phosphorylated at serine 780. A
similar result was obtained using an antibody against RB that has been
phosphorylated at threonine 373, a site that is preferentially targeted
by cyclin E- and cyclin A-dependent kinases (40). Again,
the antibody recognized the RB present in crude extracts but did not
react with the RB that cofractionates with TFIIIB (Fig. 7D).
We also tested antibodies that are specific for RB phosphorylated at
serine 795, serines 807 and 811, or threonine 252; each reacted with RB
in a crude extract but did not recognize the RB associated with
TFIIIB.4 These results suggest strongly that it is only the
underphosphorylated form of RB that cofractionates with TFIIIB, despite
the abundance of hyperphosphorylated RB in the crude extracts used as
starting material for chromatography.
Overexpression of Cyclin D-CDK4 and Cyclin E-CDK2 Stimulates pol
III Transcription in Vivo--
The above results provide evidence that
the RB that interacts with TFIIIB is not phosphorylated at sites that
are targeted by the cyclin D- and cyclin E-dependent
kinases. This is consistent with the data in Fig. 7A, which
suggest that phosphorylation of RB by these kinases may prevent it from
binding to TFIIIB. We therefore investigated how the expression of a
transfected VA1 gene would respond in vivo to the
overexpression of cyclin-dependent kinases. Transfecting
3T3 cells with expression vectors encoding cyclin D1 and its associated
kinase CDK4 resulted in a slight increase in VA1 transcription by pol
III (Fig. 8A). In contrast, cyclin E and its associated kinase CDK2 produced little or no effect
when tested in parallel. However, coexpression of cyclin D1-CDK4 with
cyclin E-CDK2 resulted in a dramatic activation of VA1. This
stimulation can be blocked by the coexpression of p16, a specific
inhibitor of cyclin D-dependent kinases.
The substantial response obtained when cyclin D1-CDK4 is combined with
cyclin E-CDK2 is unlikely to be an indirect consequence of accelerated
passage through the G1/S transition, since cyclin D1 alone
or cyclin E alone is sufficient to shorten G1 phase when overexpressed in rodent fibroblasts (18, 41, 42). A more direct effect
is consistent with the observation that TFIIIB only associates with the
underphosphorylated form of RB (Fig. 7). To investigate further the
idea that the CDKs can activate pol III transcription by overcoming
repression by RB, we made use of an SV40-transformed derivative of the
3T3 line, called SV3T3 Cl38. These SV3T3 cells express the large T
antigen of SV40, which binds and neutralizes RB (43, 44). We have shown
recently that the interaction between RB and TFIIIB is 3- or 4-fold
diminished in these cells, although not completely abolished (45). When
cyclin D1-CDK4 and cyclin E-CDK2 were cotransfected into the SV3T3
cells, VA1 transcription increased by less than 3-fold, a much weaker response than the 11-fold activation obtained in the parental 3T3 cells
(Fig. 8B). Although several explanations could account for
this difference, the data are consistent with the possibility that the
cyclin-CDKs stimulate pol III by overcoming the effect of RB on TFIIIB,
since most RB is inactive in SV3T3 Cl38 cells. The G1
cyclins also had little effect on VA1 transcription when they were
transfected into a 3T3 cell line that was transformed with the E7
oncoprotein of human papillomavirus, which binds and neutralizes
RB.5
Overexpression of protein kinases can sometimes result in reduced
substrate specificity. Indeed, at higher levels of transfection we
observed activation of VA1 by the cyclin E-CDK2 pair
alone.5 Clear evidence that some specificity is maintained
in the overexpression experiments shown in Fig. 8A is
provided by the fact that VA1 is activated by cyclin D1-CDK4 but not by
cyclin E-CDK2. Nevertheless, we wanted to test whether endogenous CDK4
and/or CDK6 are involved in controlling pol III transcription when they
are present at physiological concentrations within the cell. To address
this, we employed a ribozyme that cleaves p16 mRNA specifically at
nucleotide 89, just after the translation start site (30). 3T3 cells
were transfected with a vector encoding this ribozyme or with the empty vector, and pol III transcription of a cotransfected VA1 gene was
monitored by primer extension. The anti-p16 ribozyme was reproducibly found to increase the level of VA1 expression (Fig. 8C).
This is consistent with the stimulation obtained with cyclin D1-CDK4 and the ability of p16 to block this effect when it is overexpressed. These data support the contention that the endogenous p16/CDK/RB pathway is involved in regulating pol III activity in
vivo.
p130 Contributes to the Serum Response of pol III
Transcription--
Although the down-regulation of pol III following
serum withdrawal is substantially compromised in RB knockout
fibroblasts, some decrease is nevertheless observed (Fig. 5). This
indicates that the serum responsiveness of pol III activity is not
mediated solely by RB. Recent work has shown that the RB-related pocket proteins p107 and p130 can bind to TFIIIB and repress pol III transcription both in vitro and in vivo (7). That
study demonstrated that the effect on pol III activity of deleting p107
and p130 is most pronounced in quiescent cells; thus, p107/p130 double knockout fibroblasts show much less of a decrease in B2 transcript levels after serum deprivation than the matched cells expressing a full
complement of pocket proteins (7). These observations suggest that p107
and/or p130 play a significant role in suppressing pol III
transcription during G0 phase. Relatively little p107 is
found in serum-starved fibroblasts, but p130 is much more abundant (3,
26, 46, 47). Not only does mitogenic stimulation cause the level of
p130 to decrease, but it also becomes inactivated at the
G1/S transition through hyperphosphorylation by the cyclin D- and cyclin E-dependent kinases (3, 26, 47). This
phosphorylation can be seen clearly in Fig.
9A, where endogenous p130 is
immunoprecipitated from cells that are metabolically labeled with
[32P]orthophosphate. Although Western blotting reveals
that p130 is present in G0, G1, and S phases
(bottom panel), autoradiography shows that p130
only becomes phosphorylated once the cells have passed the
G1/S transition (upper panel). We
therefore investigated whether the high levels of active
unphosphorylated p130 present during G0 and early
G1 phases might contribute to the repression of pol III
transcription.
Coprecipitation experiments were carried out to monitor the interaction
between p130 and TFIIIB. An antiserum against p130 was used to
immunoprecipitate proteins from 3T3 cells that had been starved in low
serum or starved and then refed with 20% serum for various times prior
to harvesting; the precipitated material was then probed by Western
blotting for the BRF subunit of TFIIIB and for p130 (Fig.
9B, upper and lower panels,
respectively). Fig. 9C shows quantitation of this and a
similar experiment, in which the amount of coprecipitated BRF at each
time point has been normalized to the total amount of p130 in each
immunoprecipitation. A substantial amount of BRF coprecipitated with
p130 from the quiescent cells. This reflects a specific interaction,
since BRF was not coprecipitated with a control antiserum against
TAFI48 (lane 1). The association
between p130 and TFIIIB was maintained during early G1
phase, being undiminished 6 h after mitogenic stimulation, but had
decreased by late G1 and early S phase, 12 and 15 h
after refeeding, respectively. This dissociation correlated with the
hyperphosphorylation of p130, as indicated by phosphate labeling and a
decrease in its electrophoretic mobility (Fig. 9, A and
B, lower panel). Thus, the interaction
between TFIIIB and p130 is maximal during the G0 and early
G1 phases, when pol III transcription is repressed. We
estimate that p130 is bound to 29-34% of TFIIIB in extracts of
serum-starved cells, but this value falls to 4-7% once the cells have
entered S phase. These observations, combined with the previous
knockout data, suggest that p130 may make a significant contribution
toward suppressing pol III transcription in quiescent fibroblasts.
The data suggest that the related pocket proteins RB and p130 play
a major role in down-regulating pol III transcription when untransformed murine fibroblasts withdraw from the cell cycle following
serum deprivation. They appear to achieve this control by interacting
with TFIIIB, which leads to its repression (5-7). When quiescent cells
resume cycling in response to mitogens, RB and p130 are inactivated
through hyperphosphorylation by the cyclin D- and cyclin
E-dependent kinases in mid- to late G1 phase,
when pol III activity increases substantially. Overexpression of
cyclins D1 and E together hyperphosphorylates the pocket proteins and elicits a dramatic increase in the expression of a transfected pol III
template. Pol III transcription can also be stimulated in
vivo by depletion of endogenous p16, a specific inhibitor of cyclin D-dependent kinases. Only underphosphorylated RB
associates with TFIIIB, and the interaction is lost when RB is
phosphorylated at residues targeted by the cyclin D- or cyclin
E-dependent kinases. The data are all consistent with a
simple model in which pol III transcription is restrained in
growth-arrested fibroblasts because TFIIIB is bound and repressed by RB
and p130; mitogenic stimulation activates cyclin-dependent
kinases that phosphorylate the pocket proteins during late
G1 phase, causing them to dissociate from TFIIIB and
thereby allowing a surge in the level of pol III transcription.
Our data allow, for the first time, a continuous pathway to be traced
from the growth factors at the cell surface to the pol III machinery in
the nucleus. Ras-mediated signaling pathways are responsible for
inducing the expression of cyclin D1 and for promoting its assembly
into functional complexes with CDK4 and CDK6 (48-51). This involves
the sequential action of Raf1, the mitogen-activated protein
kinase/extracellular signal-regulated kinase kinases, and the
extracellular signal-regulated protein kinases (48-51). Ras also
controls the nuclear accumulation and proteolytic turnover of cyclin D1
through a distinct signaling pathway involving
phosphatidylinositol-3-OH kinase, protein kinase B (Akt), and glycogen
synthase kinase-3 Previous studies have shown that TFIIIC2 is down-regulated when HeLa
cells are cultured under low serum conditions (22, 23). This correlates
with a specific reduction in the abundance of its second largest
subunit TFIIIC Although we have identified the pocket proteins as key players in
coordinating pol III activity with growth factor availability, it is
extremely likely that additional control mechanisms contribute to the
overall effect. Some stimulation of pol III is observed at early time
points, prior to the major increase that occurs at the R point (19, 20,
35). It is not yet clear what is responsible for this rapid and limited
initial activation. An obvious possibility is that one or more of the
pol III transcription factors, such as TFIIIB or TFIIIC, is itself a
direct target for phosphorylation in response to mitogens. All five
subunits of TFIIIC2 are phosphorylated in HeLa cells (55), although it
is unknown whether this is constitutive or regulated, and the kinases responsible have yet to be identified. In Saccharomyces
cerevisiae, the TOR signaling pathway is involved in coordinating
pol III activity with nutrient availability (56). Casein kinase II has been shown to regulate TFIIIB in the same organism (57, 58). It
therefore seems plausible that mammalian TOR and/or casein kinase II
are also involved in controlling pol III transcription in higher
organisms. We are currently investigating these possibilities. Given
the complex mix of mitogens that is present in serum, the full response
is likely to involve multiple pathways that may feed into several
components of the pol III machinery.
An increased rate of protein synthesis is an essential feature of the
mitogenic response. Conversely, a 50% reduction in translation rate
causes cells to withdraw from cycle and quiesce (59). The availability
of tRNA and rRNA is clearly an important determinant of protein
synthetic capacity. Accordingly, growth factors induce a coordinate
induction of tRNA, rRNA, ribosomal proteins, and translation factors,
such that the rate of protein synthesis has increased substantially by
the time cells reach S phase (19, 20, 60, 61). Indeed, cells are unable
to enter S phase and duplicate their chromosomes until a sufficient
level of protein accumulation has been achieved (62, 63). Many of the
genes encoding DNA replication enzymes contain binding sites for the transcription factor E2F, which is subject to repression by RB and its
relatives during G0 and early G1 phase (64,
65). We have presented evidence that the RB family also inhibits pol
III transcription in growth-arrested fibroblasts. Inactivation of the
pocket proteins following serum stimulation will therefore result in
two major components of the mitogenic response, induction of the DNA
replication apparatus and elevated rates of synthesis of tRNA and 5 S rRNA.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice display elevated TFIIIB activity relative to
fibroblasts derived from their wild-type siblings (6). These results
establish TFIIIB as a bona fide target for repression by RB.
Similar approaches have shown that TFIIIB is also bound and repressed
by the RB-related proteins p107 and p130 (7).
(23). However, HeLa cells continue to grow
actively under the low serum conditions used in these studies (22) and may not provide a clear indication of how pol III is regulated during
exit from the cell cycle. We have therefore investigated the regulation
of pol III transcription during the transition between resting and
growing states in untransformed fibroblasts. When such cells are
stimulated to resume cycling, the major increase in tRNA synthesis
occurs during the G1/S transition (19, 20). Since this
coincides with the hyperphosphorylation of RB by
cyclin-dependent kinases, we examined the possibility that
the increase in pol III transcription that accompanies cell cycle
reentry involves a release of TFIIIB from interaction with RB.
Immunoprecipitation analyses provide evidence that this is the case; RB
associates with TFIIIB during G0 and early G1
phases, but this interaction is substantially diminished after cells
have passed the R point. The dissociation of RB from TFIIIB coincides
with an increase in pol III activity. Only the underphosphorylated form
of RB associates with TFIIIB. This suggests that TFIIIB is released
from repression by RB at the G1/S transition due to
hyperphosphorylation of the latter by the cyclin-dependent
kinases. Indeed, overexpression of cyclins D and E activates pol III
transcription. We also demonstrate that RB knockout cells are
compromised in their ability to down-regulate pol III following serum
deprivation. In addition, the RB-related pocket protein p130 is shown
to interact with TFIIIB during G0 and early G1
phase and contributes to the repression of pol III in serum-starved
cells. We conclude that RB, p130, and the cyclin-dependent kinases play a major role in controlling pol III transcription during
the switch between growth and quiescence.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, 1 mM sodium
orthovanadate, 10 mM sodium phosphate, 1 mM
phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 1.0 µg/ml
trypsin inhibitor, 0.5 µg/ml aprotinin, 40 µg/ml bestatin),
solubilized by incubation for 1 h at 4 °C, and centrifuged for
15 min at 4 °C.
, SI-1 against TFIIB (Santa Cruz Biotechnology), M-19 (Santa
Cruz Biotechnology) against TAFI48, SL30 against TBP (32),
and 128 against BRF (24, 33). Antibodies against RB that has been
phosphorylated at specific sites (34) were obtained from New England
Biolabs. Western immunoblot analysis was performed as described
previously (31).
primers (5'-CCAGAAGGGGTCTCAAAAGTCC-3' and 5'-CTTTCTTCAGAGATGTCAAAGG-3') to give a 303-base pair product, or ARPP
P0 primers (5'-GACCTGGAAGTCCAACTACTTC-3' and
5'-TGAGGTCCTCCTTGGTGAACAC-3') to give a 268-base pair product.
Amplification reactions contained 0.5 units of Taq DNA
Polymerase (Promega) in a total volume of 1× Taq DNA
polymerase buffer (Promega) containing 1.5 mM
MgCl2 and a 0.2 mM concentration of each dNTP.
PCR was performed under the following cycling parameters: 1) TFIIIC
,
94 °C for 3 min, six cycles of 95 °C for 1 min, 66 °C for
40 s, and 72 °C for 40 s; 22 cycles of 95 °C for 1 min,
62 °C for 40 s, and 72 °C for 40 s; 72 °C for 5 min;
2) ARPP P0, 95 °C for 2 min, 25 cycles of 95 °C 1 min, 58 °C
for 30 s, and 72 °C for 1 min; 72 °C for 3 min.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Pol III activity in 3T3 cells is sensitive to
the availability of serum. A, flow cytometric analyses
showing the relative DNA contents of 3T3 cells cultured for 24 h
in the absence of serum and then stimulated with 20% serum for the
indicated times prior to harvesting. Below are shown the
relative proportions of G0/G1, S, and
G2/M phase cells in each population. B, relative
levels of [3H] thymidine incorporation into newly
synthesized DNA of 3T3 cells cultured for 24 h in the absence of
serum and then stimulated with 20% serum for the indicated times prior
to harvesting. C, Northern blot analysis of total RNA (10 µg) extracted from 3T3 cells cultured in the absence of serum for
24 h (lanes 1 and 10) or cultured
without serum for 24 h and then stimulated with 20% serum for 1, 6, 9, 12, 15, 18, 21, or 24 h (lanes 2-9,
respectively). The upper panel shows the blot
probed with a B2 gene, and the lower panel shows
the same blot that has been stripped and reprobed with the ARPP P0
gene.
Remains Relatively Constant in
Growth-arrested 3T3 Cells--
To begin to investigate the mechanism
responsible for the growth control of pol III transcription in Balb/c
3T3 fibroblasts, we prepared whole cell extracts after various periods
of culture in 10% serum or serum-free medium. Although little or no
apoptosis was detected after 24 or 48 h without serum, flow
cytometry suggested that a fraction of the cells undergo apoptosis
after 72 h in serum-free medium.2 Extracts of
fibroblasts maintained without serum for 24 h or more were found
to transcribe the adenovirus VA1 gene significantly less actively than
extracts prepared from proliferating cells that had been cultured in
10% serum (Fig. 2A). Similar
results were obtained with other pol III templates, including tRNA, 5 S
rRNA, and U6 small nuclear RNA genes (Fig. 2A). The extracts therefore mimic the serum-responsiveness of pol III transcription that
is observed in vivo.
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Fig. 2.
Down-regulation of pol III activity in
serum-starved 3T3 cells is not due to a decrease in the abundance of
TFIIIC . A, plasmid templates
pVA1, pHu5S3.1, pLeu, and pU6/Hae/RA2 (500 ng) were transcribed using
15 µg of whole cell extract prepared from 3T3 cells that had been
grown continuously in 10% serum (lane 1) or
cultured without serum for 24, 48, or 72 h (lanes
2-4, respectively). B, partially purified TFIIIC
(10 µg of HeLa PC-C fraction; lane 1) and whole
cell extract (100 µg) prepared from 3T3 cells that had been grown
continuously in 10% serum (lanes 2 and
6) or cultured without serum for 24, 48, or 72 h
(lanes 3-5, respectively) were resolved on an
SDS-7.8% polyacrylamide gel and then analyzed by Western
immunoblotting with monoclonal antibody clone 46 against TFIIIC
.
C, cDNAs generated by reverse transcription of 3 µg of
RNA from 3T3 cells cultured without serum for 24 h
(lanes 1 and 11) or cultured in 0.5%
serum for 24 h and then stimulated with 20% serum for 1, 3, 6, 9, 12, 15, 18, 21, or 24 h (lanes 2-10,
respectively) were PCR-amplified using TFIIIC
(top
panel) and ARPP P0 (bottom panel)
primers. Amplification products were resolved on a 2% agarose
gel.
subunit of TFIIIC2. If this is true in untransformed 3T3 cells,
then it may account for the down-regulation of pol III transcription
under quiescent conditions. To address this possibility, we carried out
Western blots with extracts to test whether the decrease in pol III
transcription correlated with a down-regulation of TFIIIC
. However,
little or no change was detected in the level of TFIIIC
when growing
and arrested 3T3 cells were compared (Fig. 2B). Indeed,
TFIIIC
levels were maintained even after culture for 72 h
without serum. This result was confirmed using two additional antisera
raised against different regions of
TFIIIC
.3 Reverse
transcriptase-PCR analysis was employed to compare the levels of the
mRNA encoding TFIIIC
, as an independent method to investigate
the expression of this essential component of TFIIIC2. This approach
also provided no evidence that TFIIIC
is sensitive to serum
availability (Fig. 2C). We conclude that changes in the abundance of TFIIIC
are unlikely to be responsible for regulating pol III transcription in growth-arrested Balb/c 3T3 cells.
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Fig. 3.
TFIIIB activity is limiting for pol III
transcription in 3T3 cell extracts and decreases in response to serum
deprivation. A, plasmid pVA1 (500 ng) was transcribed
using whole cell extract (10 µg) prepared from 3T3 cells that had
been grown continuously in 10% serum (lanes
1-6) or cultured without serum for 72 h
(lanes 7-13). Extracts were supplemented with
1.5 (lanes 2 and 8), 3 (lanes 3 and 9), or 4.5 µl
(lanes 4 and 10) of the CHep1.0
fraction containing active TFIIIC and pol III or with 1.5 (lane 11), 3 (lanes 5 and
12), or 4.5 µl (lanes 6 and
13) of the A25(0.15) fraction containing active TFIIIB.
B, pVA1 (500 ng) was transcribed using 2 µl of CHep1.0
fraction and 1 µl of recombinant TBP supplemented with 10 µg of
heat-treated extract prepared from 3T3 cells that had been grown
continuously in 10% serum (lanes 1 and
5) or cultured without serum for 24, 48, or 72 h
(lanes 2-4, respectively). Heat treatment was at
47 °C for 15 min.
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Fig. 4.
Down-regulation of pol III activity in
serum-starved 3T3 cells is not due to a decrease in the abundance of
the TFIIIB subunits TBP and BRF. A, partially purified
TFIIIB (10 µg of PC-B fraction; lane 1) and
whole cell extract (100 µg) prepared from 3T3 cells that had been
grown continuously in 10% serum (lane 2) or
cultured without serum for 24, 48, or 72 h (lanes
3-5, respectively) were resolved on an SDS-7.8%
polyacrylamide gel and then analyzed by Western immunoblotting with
antibody 128 against BRF. B, partially purified TFIIIB (10 µg of PC-B fraction; lane 1) and whole cell
extract (100 µg) prepared from 3T3 cells that had been grown
continuously in 10% serum (lane 2) or cultured
without serum for 24, 48 or 72 h (lanes
3-5, respectively) were resolved on an SDS-7.8%
polyacrylamide gel and then analyzed by Western immunoblotting with
monoclonal antibody SL30 against TBP. C, whole cell extract
(100 µg) prepared from 3T3 cells that had been grown continuously in
10% serum (lane 1) or cultured without serum for
24, 48, or 72 h (lanes 2-4, respectively)
were resolved on an SDS-7.8% polyacrylamide gel and then analyzed by
Western immunoblotting with antibody SI-1 against TFIIB.
/
cells when compared with the wild-type cells (Fig.
5A, upper panel). Transfer of the Rb+/+ cells
into 0.5% serum results in a 2.4-fold decrease in the level of tRNA,
as expected from previous studies (19, 20, 25). However, when the
Rb
/
cells are transferred to low
serum, the abundance of tRNA shows only a 1.3-fold decrease. Indeed,
quiescent Rb
/
cells maintain a
level of tRNA expression that is close to that seen in actively growing
wild-type cells. A similar pattern is shown by the pol III transcripts
derived from B2 genes, which drop by 5.5-fold when wild-type cells are
deprived of serum but show only a 1.4-fold decrease when the RB
knockout cells are treated in the same way (Fig. 5A,
middle panel). These effects are specific, since
the pol II transcript encoding ARPP P0 is not deregulated in the RB
knockout cells (Fig. 5A, bottom
panel).
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Fig. 5.
RB knockout fibroblasts are compromised in
their ability to down-regulate pol III activity under low serum
conditions. A, Northern blot analysis of total RNA (10 µg) extracted from fibroblasts derived from
Rb /
mice (lanes
1 and 2) or matched Rb+/+
mice (lanes 3 and 4) that were
actively growing in 20% serum (lanes 2 and
4) or cultured for 24 h in 0.5% serum
(lanes 1 and 3). The blot was probed
with a tRNALeu gene (upper panel), a
B2 gene (middle panel), and an ARPP P0 gene
(bottom panel). B, nuclear run-on
assays were carried out in the presence of 1 µg/ml
-amanitin with
nuclei harvested from fibroblasts derived from
Rb+/+ mice (lanes 1 and
2) or matched Rb
/
mice (lanes 3 and 4) that were
actively growing in 20% serum (lanes 2 and
4) or cultured for 24 h in 0.5% serum
(lanes 1 and 3). Production of tRNA
was quantitated by a PhosphorImager (Molecular Dynamics, Inc.,
Sunnyvale, CA) and normalized against nonspecific background produced
in the same reaction; values shown represent the mean of two
experiments.
/
cells (Fig. 5B).
The synthesis of tRNA decreased by 2.1-fold when wild-type fibroblasts
were cultured in 0.5% serum. In contrast, only a 1.2-fold decrease was
observed in tRNA gene transcription when the
Rb
/
cells were treated in the
same way. In keeping with the Northern blots, the nuclear run-ons
showed that tRNA synthesis is maintained in serum-starved
Rb
/
cells at a level very close
to what is observed in the actively proliferating wild-type cells.
These data provide clear genetic evidence that RB plays a major role in
controlling pol III transcription during the switch between growth and
quiescence. It is nevertheless apparent that RB is not solely
responsible for this control, since some down-regulation is still
observed when Rb
/
cells are
cultured in low serum; we shall return to this point later.
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Fig. 6.
The interaction between RB and TFIIIB is
maximal during G0 and early G1 phase.
A, anti-RB antibody C-15 (lanes 1-6)
and anti-TAFI48 antibody M-19 (lane
7) were used to immunoprecipitate material from whole cell
extracts (150 µg) of 3T3 cells cultured without serum for 24 h
(lanes 1 and 7) or cultured without
serum for 24 h and then stimulated with 20% serum for 3 (lane 2), 6 (lane 3), 9 (lane 4), 12 (lane 5), or
15 h (lane 6). Immunoprecipitates were
resolved on an SDS-7.8% polyacrylamide gel and then analyzed by
Western blotting with anti-BRF antiserum 128 (upper
panel) and anti-RB antiserum C-15 (lower
panel). B, quantitation of the level of
interaction between TFIIIB and RB following mitogenic stimulation.
Densitometry was used to compare the relative amounts of BRF
coprecipitated, and this was normalized against the amounts of RB in
the same immunoprecipitate. Values are expressed in arbitrary units,
and the value obtained for unstimulated (G0) cells is
designated 1. The values shown represent the mean of three experiments,
including the one illustrated in Fig. 6A. C,
whole cell extracts (50 µg) of 3T3 cells cultured without serum for
24 h (lane 1) or cultured without serum for
24 h and then stimulated with 20% serum for 3 (lane
2), 6 (lane 3), 9 (lane
4), 12 (lane 5), or 15 h
(lane 6) were resolved on an SDS-7.8%
polyacrylamide gel and then analyzed by Western blotting with antiserum
that specifically recognizes RB phosphorylated at serine 780.
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Fig. 7.
TFIIIB associates with underphosphorylated RB
but not with hyperphosphorylated RB. A,
35S-labeled RB was synthesized in vitro using a
reticulocyte lysate. An aliquot of this material was then incubated
with a mixture of baculovirus-expressed recombinant cyclin D1-CDK4,
cyclin E-CDK2, and cyclin A-CDK2. Lysates containing either
unphosphorylated RB (lanes 3 and 4) or
hyperphosphorylated RB (lane 5) were then
incubated with 3T3 cell extract (150 µg) and subjected to
immunoprecipitation (IP) with either preimmune serum
(lane 3) or anti-BRF antiserum 128 (lanes 4 and 5). Proteins retained
after extensive washing were resolved on an SDS-7.8% polyacrylamide
gel and then visualized by autoradiography. Lanes
1 and 2 show 10% of the input reticulocyte
lysate containing either unphosphorylated or hyperphosphorylated RB,
respectively. B, nuclear extract (62 µg) prepared from
actively growing HeLa cells (lane 1) and TFIIIB
(A25(0.15) fraction, 0.8 µg) partially purified from actively growing
HeLa cells (lane 2) were resolved on an SDS-7.8%
polyacrylamide gel and then analyzed by Western immunoblotting with
antibody G3-245 against RB. C, nuclear extract (60 µg)
prepared from actively growing HeLa cells (lane
1) and TFIIIB (A25(0.15) fraction, 2.4 µg) partially
purified from actively growing HeLa cells (lane
2) was resolved on an SDS-7.8% polyacrylamide gel and then
analyzed by Western immunoblotting with antibody against RB
phosphorylated at serine 780. D, as for C, except
that the antibody used was against RB phosphorylated at threonine
373.
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Fig. 8.
Overexpression of cyclin D1-CDK4 together
with cyclin E-CDK2 activates pol III transcription in
vivo. A, 3T3 cells growing in 10% serum
were transfected with pVA1 (2 µg, all lanes),
pCAT (2 µg, all lanes), pRc-CMV vector (6 µg
in lane 1, 4 µg in lanes
2 and 3, 3 µg in lane 4,
and 1.5 µg in lane 5), Rc-CDK4 (1 µg in
lanes 2, 4, and 5),
Rc-cycD1 (1 µg in lane 2, 0.5 µg in
lanes 4 and 5), Rc-CDK2 (1 µg in
lanes 3-5), Rc-cycE (1 µg in lane
3, 0.5 µg in lanes 4 and
5), and pCMVp16 (1.5 µg in lane 5).
VA1 and CAT RNA levels were assayed by primer extension and then
quantitated using a PhosphorImager. Values shown are for VA1 expression
after normalization to the levels of CAT RNA to correct for
transfection efficiency; they are given relative to the value obtained
with pRc-CMV vector alone (designated 1.0) and represent the mean of
two experiments. B, 3T3 cells (lane 1)
and SV3T3 Cl38 cells (lane 2) growing in 10%
serum were transfected with pVA1 (2 µg), pCAT (2 µg), and pRc-CMV
vector (6 µg) alone or pRc-CMV vector (3 µg), Rc-CDK4 (1 µg),
Rc-cycD1 (0.5 µg), Rc-CDK2 (1 µg), and Rc-cycE (0.5 µg). VA1 and
CAT RNA levels were assayed by primer extension and then quantitated
using a PhosphorImager. VA1 expression was then normalized to the level
of CAT RNA to correct for transfection efficiency. The values shown
give the increase in normalized VA1 expression in response to
CDK4-cyclin D1 and CDK2-cyclin E. C, 3T3 cells growing in
10% serum were transfected with pVA1 (2 µg), pCAT (2 µg), and 2 µg of pX vector (lane 1) or Rz 89-12 anti-p16
ribozyme (lane 2). VA1 and CAT RNA levels were
assayed by primer extension and then quantitated using a
PhosphorImager. Values shown are for VA1 expression after normalization
to the levels of CAT RNA to correct for transfection efficiency; they
are given relative to the value obtained with pX vector and represent
the mean of four experiments.
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Fig. 9.
p130 interacts with TFIIIB in growth-arrested
fibroblasts but dissociates during the G1/S
transition. A, 3T3 cells were cultured without serum
for 36 h and then incubated in phosphate-free DMEM for 15 h
in the presence of [32P]orthophosphate. Cells were either
maintained without serum (lane 1) or stimulated
with 10% serum for 3 h (lane 2) or 15 h (lanes 3 and 4). They were then
harvested and immunoprecipitated with antibody C-20 against p130
(lanes 1-3) or, as negative control, with
antibody Y-11 against the hemagglutinin tag (HA;
lane 4). Immunoprecipitates were resolved on an
SDS-7.8% polyacrylamide gel, transferred to nitrocellulose and
subjected to autoradiography (upper panel). The
membrane was then analyzed by Western blotting with antibody C-20
against p130 (lower panel). B,
anti-TAFI48 antibody M-19 (lane 1)
and anti-p130 antibody C-20 (lanes 2-7) were
used to immunoprecipitate material from whole cell extracts (150 µg)
of 3T3 cells cultured without serum for 36 h (lanes
1 and 2) or cultured without serum for 24 h
and then stimulated with 20% serum for the indicated times.
Immunoprecipitates were resolved on an SDS-7.8% polyacrylamide gel and
then analyzed by Western blotting with antibody 128 against BRF
(upper panel) and antibody C-20 against p130
(lower panel). C, quantitation of the
level of interaction between TFIIIB and p130 following mitogenic
stimulation. Densitometry was used to compare the relative amounts of
BRF coprecipitated, and this was normalized against the relative
amounts of p130 in the same immunoprecipitates. Values are expressed in
arbitrary units, and the value obtained for unstimulated
(G0) cells is designated 1. The values shown represent the
mean of two experiments, including the one illustrated in
B.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(52). Thus, when mitogens activate Ras via their
cell surface receptors, one net result is the nuclear accumulation of
functional complexes containing cyclin D1 bound to activated CDK4 and
CDK6. These, in turn, phosphorylate RB at specific sites, inducing
conformational rearrangements that allow the remaining phosphoacceptor
residues to be phosphorylated by cyclin E-CDK2 (53). As we have shown,
the hyperphosphorylated RB dissociates from TFIIIB, thereby
derepressing it and allowing a surge in pol III transcriptional
activity. It is clear that Ras plays a key role in this process, which
is consistent with a previous study that showed that constitutively
active Ras will stimulate pol III transcription in Rat-1 fibroblasts
(54). We have confirmed that this is also true of the murine Balb/c 3T3 fibroblast system that was employed in the current study.2
Furthermore, inhibitors of Ras, mitogen-activated protein
kinase/extracellular signal-regulated kinase kinases, or
phosphatidylinositol-3-OH kinase can all compromise the mitogenic
response of pol III in Balb/c 3T3 cells.2 We therefore
believe that growth factors can regulate class III genes in this cell
type via TFIIIB, RB, cyclin D, and the signal transduction pathways
that connect these to Ras.
(23). We have found no evidence for such an effect in
Balb/c 3T3 cells, where the level of TFIIIC
polypeptide remains
undiminished after 3 days of serum deprivation. Expression of the
mRNA encoding TFIIIC
is also maintained under low serum
conditions. Several possible explanations could account for the
distinct behavior of TFIIIC
in these two systems. HeLa cells are
highly transformed and were derived from human cervical epithelium,
whereas Balb/c 3T3 cells are untransformed murine fibroblasts; any one
(or more) of these differences between the two cell types might be
responsible for the distinct response of TFIIIC
to serum
deprivation. Given the malignant nature of HeLa cells and their
continued growth under low serum conditions, the 3T3 cell system is
much more likely to reflect a normal physiological response to mitogen withdrawal.
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ACKNOWLEDGEMENTS |
---|
We thank Bill Cushley for help with flow cytometry, Sibylle Mittnacht for advice and mouse embryonic fibroblasts, Michael Strauss and Jesper Nylandsted for Rz 89-12 and pCMVp16, and Fred Dick for ARPP P0.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Research Career Development Fellowship 055409 (to P. H. S.) from the Wellcome Trust, by Biotechnology and Biological Sciences Research Council Grant CO5766 (to R. J. W.), and by Association for International Cancer Research Grant 98-46 (to R. J. W.).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.
A Wellcome Trust Research Fellow.
§ A Jenner Research Fellow of the Lister Institute of Preventive Medicine. To whom correspondence should be addressed. Tel.: 44-141-330-4628; Fax: 44-141-330-4620; E-mail: rwhite@udcf.gla.ac.uk.
Published, JBC Papers in Press, October 9, 2000, DOI 10.1074/jbc.M005417200
2 P. H. Scott, unpublished data.
3 H. M. Alzuherri, unpublished data.
4 R. J. White, unpublished data.
5 C. A. Cairns, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: pol, RNA polymerase; ARPP P0, acidic ribosomal phosphoprotein P0; CDK, cyclin-dependent kinase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; TF, transcription factor; TOR, target of rapamycin; CMV, cytomegalovirus; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; TBP, TATA-binding protein.
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