Institute of Medical Biochemistry, University of Oslo, PO Box 1112, Blindern, N-0317, Oslo, Norway
* Author for correspondence (e-mail: h.k.blomhoff{at}basalmed.uio.no )
Accepted 3 December 2001
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Summary |
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The transient dephosphorylation of pRB could be explained by the transient decrease in the activities of the pRB-specific kinases, but to understand why pRB became only partially rephosphorylated, despite fully activated kinases, we postulated that cAMP could activate a pRB-directed phosphatase. It was therefore interesting to find that the phosphatase inhibitor, tautomycin, was able to abolish the forskolin-mediated dephosphorylation of pRB, without increasing the activities of the pRB-specific kinases.
To understand how Reh cells expressing hyperphosphorylated forms of pRB can remain arrested in G1, we used three different methods to test for the ability of pRB to form functional complexes with the family of E2F transcription factors. As expected, we observed an increased complex formation between E2F-1, E2F-4 and pRB after 2 hours when pRB was in its most dephosphorylated state. Suprisingly, however, prolonged treatment with forskolin, which induced partial rephosphorylation of pRB, in fact further increased the complex formation between the E2Fs and pRB, and this also resulted in reduced E2F-promoter activity in vivo. These data imply that in Reh cells, partially phosphorylated forms of pRB retain the ability to inhibit E2F-promoter activity, and thereby prevent cells from entering into S-phase.
Key words: Forskolin, G1-arrest, pRB, E2F
![]() |
Introduction |
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The retinoblastoma protein is a member of a family of pocket proteins,
including p107 (Ewen et al.,
1991) and p130 (Hannon et al.,
1993
; Mayol et al.,
1993
). Together with pRB itself, these proteins regulate the
activity of the E2F family of transcription factors
(Dyson, 1998
). The RB protein
binds directly to E2F and represses E2F-mediated transcription, a function
that is prevented by the phosphorylation of pRB by CDKs
(Hatakeyama et al., 1994
;
Lundberg and Weinberg, 1998
).
Transfection experiments have shown that overexpression of E2Fs or CDKs
promote S-phase entry (Johnson et al.,
1993
), whereas overexpression of the pRB family of proteins leads
to G1 arrest (Goodrich et al.,
1991
; Qin et al.,
1992
). The growth-inhibitory function of pRB, p107 and p130 maps
to the domain known to involve E2F binding
(Weintraub et al., 1992
;
Sellers et al., 1995
),
suggesting that the inhibitory effects of the pRB family proteins on
proliferation are mediated by their suppressive effects on E2F-induced gene
expression.
Numerous studies have shown that inhibition of cell-cycle progression of
both normal and malignant cells frequently occurs in G1, linking this to an
inhibition of pRB phosphorylation
(Miyatake et al., 1995;
Li et al., 1997
;
Kawamata et al., 1998
). Using
normal human lymphocytes we have previously shown that increased levels of
cAMP lead to inhibition of pRB phosphorylation and arrest of cells in G1
(Naderi et al., 2000
). By
contrast, we observed that cAMP-mediated G1 arrest of a cell line derived from
an acute lymphoblastic leukaemia, Reh, was accompanied by a transient
inhibition of pRB phosphorylation
(Christoffersen et al., 1994
;
Naderi and Blomhoff, 1999
). In
these cells, cAMP, through activation of PKA, induced dephosphorylation of pRB
within 2 hours of treatment, followed by partial rephosphorylation of pRB
within the next 24 hours (Naderi and
Blomhoff, 1999
).
To investigate the mechanisms responsible for forskolin-mediated transient dephosphorylation of pRB, we examined the activity of pRB-specific kinases after forskolin treatment of Reh cells. Forskolin inhibited the activity of these kinases, the kinetics of which seemingly paralleled the dephosphorylation of pRB. This inhibition was transient, because within 72 hours of treatment the activity of these kinases was restored to the levels found in untreated cells. Interestingly, these cells, despite the presence of active CDKs, failed to phosphorylate pRB to the levels found in control cells and exhibited only a partially rephosphorylated pRB. These observations raised the following two questions that we have addressed in this study: how can pRB remain only partially rephosphorylated when the CDKs are fully active, and how can the Reh cells expressing phosphorylated forms of pRB remain permanently arrested in G1?
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Materials and Methods |
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Cell culture of Reh cells
The B-lymphoid precursor cell line Reh was originally derived from a
patient with acute lymphoblastic leukaemia
(Rosenfeld et al., 1977) and
was kindly provided by M. F. Greaves (Imperial Cancer Research Fund
Laboratories, London, UK). The cells were cultured at a density between
0.15x106 and 1.5x106 cells/ml in RPMI 1640,
supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 2 mM
glutamine, penicillin (125 U/ml) and streptomycin (125 µg/ml) at 37°C
in a humidified incubator with 5% CO2.
Assessment of cell proliferation
Cell counting was measured through determination of DNA synthesis and was
analysed by incorporation of [3H]thymidine into DNA. Cells were
cultured in microtiter plates at an initial density of 1x104
cells/0.2 ml, and were pulsed with 0.2 µCi of [3H]thymidine
(Amersham Pharmacia Biotech) for the last 20 hours of a 72 hour incubation.
The cells were then harvested and counted on a cell harvester and
scintillation counter (Topcount; Packard).
Flow cytometric analysis of cell-cycle distribution
The cell-cycle distribution of forskolin-treated Reh cells was assessed by
pulse labelling the cells with BrdU 1 hour before harvesting. The cells were
fixed in 70% ethanol before staining the cells with a FITC-conjugated
anti-BrdU antibody and PI (Ohtani,
1999). The cell-cycle distribution was analysed using a FACScan
flowcytometer (Becton Dickinson, San Jose, CA) according to the manufacturer's
procedure.
Immunoblot analysis
The expression of the different cell-cycle regulatory proteins was carried
out by immunoblot analysis as described by Naderi and Blomhoff
(Naderi and Blomhoff,
1999).
Detection of different phosphorylation forms of RB was performed by
preparing whole cell extracts as described by Naderi and Blomhoff
(Naderi and Blomhoff, 1999).
Equal amounts of lysate (50 µg) were run on a 10% SDS-PAGE and the resolved
proteins were transferred to a nitrocellulose membrane (Amersham) using a
semidry transfer cell (Bio-Rad, Hercules, CA). Unspecific sites were blocked
by incubating the membrane in blocking buffer (1xTris buffered saline
(TBS), 0.1% Tween with 5% nonfat dry milk) for 1 hour, followed by washing the
blot three times in 1xTBS containing 0.1% Tween (TBST). The immunoblot
was then incubated at 4°C overnight with primary antibody diluted 1:1000
in blocking buffer. After washing the immunoblot three times in TBST, the blot
was incubated for 1 hour at room temperature with horse radish peroxidase
(HRP)-conjugated secondary antibody diluted 1:7000 in blocking buffer. The
immunoreactive proteins were visualised with the enhanced chemiluminescence
detection system (ECL, Amersham Pharmacia Biotech) according to the
manufacturer's protocol.
Immunoprecipitation
For immunoprecipitation of E2F-1 and E2F-4, cell pellets were resuspended
in Triton X-100 lysis buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 0.1% Triton
X-100, 10 µg/ml leupeptin, 9.5 µg/ml aprotinin, 35 µg/ml
phenylmethylsulfonyl fluoride, 5 mM NaF, 0.1 mM orthovanadate, 10 mM
ß-glycerophosphate). The samples were placed on ice and vortexed at 5
minute intervals for 20 minutes. After removing the insoluble material by
centrifugation, the lysates were precleared by incubation with 25 µl of a
1:1 slurry of protein G-sepharose (Pharmacia, Sweden) for 30 minutes at
4°C. The protein content was assessed by the Bradford method (Bio-Rad).
Cell lysates (600 µg) were immunoprecipitated with the appropriate antibody
(2 µg/sample) for 2 hours at 4°C. The immunocomplexes were absorbed to
30 µl of a 1:1 slurry of protein G-sepharose for 1 hour at 4°C,
collected by centrifugation at 2,000 g for 5 minutes and
washed twice with the appropriate lysis buffer. The beads were then
resuspended in 1xSDS sample buffer and boiled, and the proteins were
subjected to SDS-PAGE and immunoblot analysis as described by Naderi and
Blomhoff (Naderi and Blomhoff,
1999).
Kinase assays
Measurements of cyclin E- and cyclin A-associated kinase activity were
performed essentially as described by Naderi and Blomhoff
(Naderi and Blomhoff, 1999),
using histone H1 as substrate.
Cyclin D3-associated kinase activity was analysed using
glutathione-S-transferase-tagged RB (amino acids 769-921), denoted GST-RB
(Santa Cruz Biotechnology, Santa Cruz, CA), as substrate. Whole cell extracts
were prepared by sonication in equal amounts of lysis buffer (50 mM HEPES pH
7.5, 50 mM NaCl, 10% glycerol, 0.1% Tween 20, 1 mM dithiothreitol (DTT), 20 mM
Na-pyrosphosphate, 50 mM NaF, 0.3 mM orthovanadate, 80 mM
ß-glycerophosphate, 10 µg/ml leupeptin, 10 µg/ml antipain, 10
µg/ml chymostain, 10 µg/ml pepstatin A, 5 µg/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride (PMSF) and 100 µg/ml Benzamidin). The
supernatant after centrifugation was precleared by incubation with 25 µl of
a 1:1 slurry of protein G-sepharose (Pharmacia, Sweden) for 30 minutes at
4°C and analysed for protein content by the Bradford method (Bio-Rad).
Cell lysate (1 mg) was immunoprecipitated with the appropriate antibody (2
µg/sample) for 2 hours at 4°C. The immunoreactive complexes were
absorbed to 30 µl of a 1:1 slurry of protein G-sepharose for 1 hour at
4°C, collected by centrifugation at 2000 g for 5 minutes.
The immunoreactive complexes were washed four times with lysis buffer and once
in 50 mM HEPES (pH 7.5) containing 1 mM DTT. The washed complexes were
resuspended in 30 µl of kinase buffer (50 mM HEPES pH 7.5, 10 mM
MgCl2, 1 mM DTT, 2.5 mM EGTA, 1 mM NaF, 0.3 mM sodium
orthovanadate, 10 mM ß-glycerophosphate, 20 µM ATP) and the kinase
reactions were initiated by adding 10 µCi of [-32P]ATP
and 2 µg of GST-RB (amino acids 769-921, Santa Cruz Biotechnology) to each
reaction. After 30 minutes at 30°C, the reactions were stopped by adding
15 µl 3xSDS sample buffer. The samples were boiled for 5 minutes and
subjected to SDS-PAGE. Following electrophoresis, gels were stained with
Coomassie blue, dried and subjected to autoradiography.
Electrophoresis mobility shift assay (EMSA)
Whole-cell extracts were prepared essentially as described by Pagano et al.
(Pagano et al., 1992);
20x106 Reh cells were collected, washed once in
phosphate-buffered saline (PBS) and resuspended in 200 µl of lysis buffer A
(20 mM HEPES pH 7.9, 0.4 M NaCl, 25% glycerol, 1 mM EDTA, 2.5 mM DTT and 1 mM
PMSF). Cells were incubated for 20 minutes on ice, frozen in liquid nitrogen
and placed at -70°C. Cell extracts were thawed on ice and the suspensions
were vigorously mixed, followed by centrifugation for 10 minutes at 13,000
g. The supernatant was collected and the protein content
measured by the Bradford method (Bio-Rad). Total lysate, 20 µg, was
incubated with approximately 0.5 ng (40,000 c.p.m.) of 32P-labelled
double-stranded oligonucleotide, containing a consensus E2F-binding site
(Santa Cruz Biotechnology), in a final volume of 30 µl. Sonicated salmon
sperm DNA (10 µg) was added as competitor DNA. Incubations were carried out
at room temperature for 20 minutes in 20 mM HEPES, 20 mM NaCl, 2 mM
MgCl2, 10% glycerol, 1 mM DTT, 1 mM PMSF and 20 µg BSA
(Lam and Watson, 1993
).
Supershift assays were performed by incubating the reaction mixture with the
appropriate antibody, on ice for 1 hour, before adding the labelled
oligonucleotide. The DNA-protein complexes were resolved on a 4% polyacrylamid
gel in 0.25xTBE (Tris borate-EDTA) buffer at 4°C, and the
protein/DNA complexes were visualised by autoradiography.
Promoter reporter assay
Exponentially growing Reh cells were transiently transfected with the
following luciferase constructs: pGL3TATAbasic-6xE2F (pGL3 containing a TATA
box and six E2F binding sites, 5'-TTTCGCGCTTAA-3', kindly provided
by Ali Fattaey, Onyx Pharmaceuticals, Richmond, CA) and a pGL3TATAbasic (an
E1b TATA box cloned into pGL3, Promega). An SV40-ßGal construct (Promega
Corp., E1081) was contransfected and used as an internal control. Transfection
was performed by electroporation at 250 V and 950 µF. The electroporated
cells were transferred to fresh medium and incubated for 20 hours before the
cells were treated with forskolin. Cells were harvested and resuspended in
reporter lysis buffer, 20 µl/million cells, (Promega Corp., E3971).
Luciferase activity was measured according to the manufacturer's protocol
(Promega Corp., E1501) and by using an LKB Wallac 1251 luminometer (LKB,
Helsinki, Finland). The ß-galactosidase activity was analysed according
to the manufacturer's protocol (Promega Corp., E2000), where the absorbance at
405 nm was measured by spectrophotometry (Labsystems Multiscan, Bichromatic).
Luciferase activity was normalized to ß-galactosidase activity.
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Results |
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|
Forskolin induces transient dephosphorylation of pRB
We have previously shown that forskolin treatment of Reh cells leads to a
rapid, but transient dephosphorylation of pRB
(Christoffersen et al., 1994;
Naderi and Blomhoff, 1999
).
Consistent with our previous results, we observed a rapid dephosphorylation of
pRB after 2 hours of forskolin treatment, followed by a rephosphorylation
notable after 24 hours (Fig.
2A). However, the rephosphorylation was only partial, as the fully
phosphorylated forms of pRB were less apparent in forskolin-treated cells.
Interestingly, pRB remained partially rephosphorylated 72 hours after addition
of forskolin (Fig. 2A), at the
time when the cells were growth-inhibited and accumulated in G1
(Fig. 1;
Table 1).
|
To identify the various migrating forms of pRB that we observed in
Fig. 2A, different controls
were run together with lysates from Reh cells treated with or without
forskolin for 2 hours (Fig.
2B). As a control for unphosphorylated pRB, we used lysates from
unstimulated normal T-lymphocytes, known to be in G0. To rule out the
possibility that the lower molecular weight form was not equivalent to the
C-terminally cleaved form of pRB seen in apoptotic cells, we included lysates
from Jurkat cells induced to undergo apoptosis by stimulation with the
anti-Fas antibody, CH11, for 4 hours (Tan
et al., 1997). As a source for fully phosphorylated form of pRB,
we used lysates from normal T-lymphocytes activated into late parts of the
cell cycle (S/G2/M) by stimulation with phytohemagglutinin-P (PHA)/ionomycin
for 50 hours. From Fig. 2B it
is evident that the lower molecular weight form of pRB, appearing rapidly
after forskolin treatment, migrates as the unphosphorylated form of pRB found
in resting T cells and not as the cleaved form of pRB.
To examine whether the partial rephosphorylation of pRB seen in
Fig. 2A was due to some sites
being permanently dephosphorylated, we performed immunoblot analysis of pRB
using different antibodies directed against specific phosphorylated amino
acids in the C-terminal region of pRB. It has been shown that several residues
in this region are involved in the regulation of E2F-binding
(Knudsen and Wang, 1997), and
that several, if not all of these sites needs to be phosphorylated to disrupt
the binding of E2F (Lundberg and Weinberg,
1998
; Brown et al.,
1999
; Harbour et al.,
1999
). We therefore selected four commercially available
antibodies directed against phosphorylated serine and threonine residues in
this region (Ser 780, Ser 795, Ser 807/811, Thr 821). As shown in
Fig. 2C, all four amino acids
were phosphorylated in continuously growing cells. Ser 780, Ser 795 and Ser
807/811 were subjected to dephosphorylation at 2 hours of forskolin treatment,
whereas only minor changes were observed in the case of Thr 821. Of note is
that the dephosphorylation of Ser 780, Ser 795 and Ser 807/811 was transient,
but the rephosphorylation of these sites was only partially restored after
prolonged forskolin treatment. Apparently, none of these sites were
permanently dephosphorylated.
Taken together, we believe that after 2 hours of forskolin treatment the pRB-population consists mainly of unphosphorylated and hypophosphorylated forms. After 48-72 hours of forskolin treatment we observe a reduction in the unphosphorylated and the most hypophosphorylated form of pRB, concomitant with an increase in the pRB-population that is rephosphorylated at several of the C-terminal residues. However, the amount of fully or hyperphosphorylated forms of pRB after 72 hours of forskolin treatment must be limited, as Fig. 2C shows that at least three of the C-terminal residues are not phosphorylated in a large portion of the pRB molecules.
Forskolin induces a transient reduction in cyclin-associated kinase
activity
To test whether the rapid dephosphorylation of pRB followed by incomplete
rephosphorylation could be explained by the regulation of the activity of
pRB-specific kinases, we analysed the effect of forskolin on various CDKs. As
shown in Fig. 3, forskolin
transiently inhibited the activity of the different cyclin-associated kinases.
The activity of cyclin D3- and cyclin E-associated kinases were reduced
threefold after 8 hours of forskolin treatment. However, the activity of these
two kinases was fully restored after 72 hours. Cyclin A-associated kinase
(Fig. 3) and CDK1 (data not
shown) exhibited an alternating pattern of activity. At 2 and 24 hours post
treatment these kinases showed a twofold reduction in activity, whereas at 8
and 72 hours post treatment, their activity was restored to control levels.
Taken together, these results suggest that the immediate dephosphorylation of
pRB by forskolin involves the reduction in the CDK kinase activity. However,
as the dephosphorylation of pRB seen in
Fig. 2A seems to be faster than
the inhibition of the overall kinase activity, this may support our findings,
presented below, that forskolin also activates a pRB-specific phosphatase.
This would also explain why we observe that pRB is only partially
rephosphorylated after 48-72 hours of forskolin treatment, despite fully
active CDKs.
|
Effect of forskolin on protein expression of G1 cyclins, CDKs and
CKIs
To understand how forskolin induces the transient inhibition of the CDK
activities, we analysed the expression of the various cyclins, CDKs and CKIs.
As shown in Fig. 4A, the
expressions of cyclin D3 and cyclin E followed the activity level of their
respective kinases. Within 2-8 hours of forskolin treatment, the level of
these cyclins was reduced compared with untreated cells, whereas after 72
hours the expression levels were resotred to the levels seen in untreated
cells. Cyclin A expression generally showed the same cycling appearance as the
activity of its associated kinase (Fig.
3). The protein levels of CDK 2, 4 and 6 were not altered during
forskolin treatment (Fig. 4A),
whereas the expression of both p21Cip1 and
p27Kip1 were transiently increased with maximum levels at
8 and 24 hours, respectively (Fig.
4B).
|
The phosphatase 1 inhibitor, tautomycin, abolishes the effect of
forskolin on RB-phosphorylation
Despite full restoration of the activity of pRB-specific kinases, Reh cells
treated with forskolin for more than 24 hours contained only partially
rephosphorylated pRB (Fig. 2).
This observation suggested that forskolin-induced dephosphorylation of pRB
also could involve activation of a protein phosphatase. To address this
possibility, we examined the effect of several known inhibitors of PP1 and
PP2A (okadaic acid, caliculin and tautomycin) on forskolin-mediated
dephosphorylation of pRB. All the inhibitors tested completely abolished the
effect of forskolin (okadaic acid and caliculin, data not shown). In
Fig. 5A, pretreatment of Reh
cells with the most specific of the PP1 inhibitors, tautomycin, prevented the
forskolin-mediated dephosphorylation of pRB observed after 4 hours.
Furthermore, tautomycin alone also induced hyperphosphorylation of pRB.
|
To exclude the possibility that the inhibitory effect of tautomycin on forskolin-mediated dephosphorylation of pRB was due to activation of the pRB-specific kinases, we measured the effect of tautomycin on the CDK activity. As shown in Fig. 5B, the cyclin-associated kinase activities were, in fact, reduced rather than activated by tautomycin, as were the expression of the cyclins also (Fig. 5C). Taken together, our results therefore suggest a role for protein phosphatase 1 or 2A, or both, in cAMP-mediated dephosphorylation of pRB.
Transient dephosphorylation of p130 and p107 by forskolin
The pocket proteins p107 and p130 also showed a transient dephosphorylation
after forskolin treatment, as did pRB. As shown in
Fig. 6, a rapid
dephosphorylation was evident after 2 hours of forskolin treatment and a
rephosphorylation appeared after 24 hours of treatment. The rephosphorylation
of p107 and p130 was only partial after 72 hours of forskolin treatment.
|
E2F-RB complex formation is increased by forskolin
Both pRB itself and the related pocket proteins, p130 and p107, exert their
growth-inhibitory effects by binding to members of the E2F/DP1 family of
transcription factors and leading to repression of transcription from
E2F-regulated promoters (Chellappan et al.,
1991; Weinberg,
1995
; Harbour and Dean,
2000
). It is generally believed that pRB binds to E2Fs in its
hypophosphorylated form, and that the binding is disrupted when pRB is
hyperphosphorylated (Knudsen and Wang,
1997
; Helin,
1998
). As we showed that pRB
(Fig. 2A), as well as p107 and
p130 (Fig. 6), was partially
rephosphorylated by prolonged treatment of forskolin, we wished to examine
whether these pocket proteins were still able to bind E2Fs. We used two
different methods to test the complex formation between E2Fs and pRB, i.e.
co-immunoprecipitation and gel retardation assay. Using the first method,
E2F-1 and E2F-4 were immunoprecipitated and the resulting immunoblots were
incubated with antibodies against pRB. As shown in
Fig. 7A, complexes between pRB
and E2F-1 or E2F-4 were noted already after 2 hours of forskolin treatment,
but interestingly, the amount of pRB bound to both E2F-1 and E2F-4 increased
with longer exposure of the cells to forskolin. As E2F-4 has also been shown
to bind to other pocket proteins, such as p130 and p107
(Beijersbergen et al., 1994
;
Ginsberg et al., 1994
;
Vairo et al., 1995
), we also
examined the complex formation between p130 or p107 and the E2F-4 proteins.
The complex formation essentially followed that of pRB-E2F-4, but the amount
of p130 or p107 associated with E2F-4 appeared to be much less than the amount
of pRB bound to E2F-4 (data not shown). No complexes between p130 or p107 and
E2F-1 were observed (data not shown). Notably, the expression levels of E2F-1
and E2F-4 remained unaffected by forskolin treatment
(Fig. 7B).
|
In the second method, we used gel retardation assay to examine E2F-complexes following treatment of the cells with forskolin. Total extracts were incubated with radioactive labelled E2F-specific oligonucleotides and the resulting complexes were separated on a native polyacrylamid gel. We detected three specific complexes (I, II and III in Fig. 8A). All three complexes were differentially regulated by forskolin, as the formation of complex III decreased upon exposure to forskolin, concomitantly with increased formation of complexes I and II.
|
The composition of the different complexes were determined by gel retardation supershift assays, using a panel of specific antibodies directed against proteins known to form complexes with E2Fs and DPs. As shown in Fig. 8B, complex III was shifted by antibodies directed against E2F-1 and E2F-4, but not by antibodies specific for pRB. This complex was, to a certain extent, also shifted by antibodies specific for DP1 (Fig. 8C), but remained unaffected by antibodies directed against p107 or p130 (Fig. 8C). This suggests that complex III consists of E2F-1/E2F-4 and DP-1. Complex II was shifted by antibodies against pRB, E2F-1, E2F-4 (Fig. 8B) and DP-1 (Fig. 8C), but not by antibodies against p107 and p130 (Fig. 8C), and therefore represents the complex between pRB and E2F-1 or E2F-4, including DP-1. Complex I, however, was shifted by antibodies against p107, p130, E2F-4 and cyclin A (Fig. 8B,C).
Taken together, the gel retardation assay showed that the complex formation between pRB and E2F-1 or E2F-4 increases after 72 hours of forskolin treatment, at the time when pRB is partially rephosphorylated (Fig. 2A). Furthermore, complexes between p107 or p130 and E2F-4 are prominent already at 2 hours of forskolin treatment, and this complex formation is further increased upon longer exposure to forskolin (72 hours). Concomitant with the increased complex formation between pRB and E2Fs, the fraction of free E2Fs bound to the E2F-consensus site is reduced.
Forskolin inhibits E2F-promoter activity in vivo
To verify that the increased complex formation between the pocket proteins
and E2Fs resulted in functional inhibition of E2F-mediated transcription, we
examined the effect of forskolin on the E2F-promoter activity in vivo using a
pGL3-Luciferase reporter construct containing a TATA-6xE2F-promoter fragment
or a basic-pGL3TATA-Luciferase construct. As shown in
Fig. 9A, the relative
luciferase activity was reduced by 10% after 2 hours of forskolin treatment,
whereas after 24 (Fig. 9B) and
72 hours (Fig. 9C) of treatment
the E2F-promoter activity was reduced by 40% and 46%, respectively. Thus, we
can conclude that the increased complex formation between the pocket proteins
and E2F in the presence of forskolin resulted in functional inhibition of
E2F-mediated transcription.
|
![]() |
Discussion |
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It has been suggested that protein phosphatase 1 (PP1) can interact with
pRB in G1 (Liu et al., 1999),
and it has been shown that PP1 is the phosphatase responsible for
dephosphorylating pRB at the entry of cells into G1 from mitosis
(Durfee et al., 1993
;
Nelson and Ludlow, 1997
). To
examine the possibility of phosphatases being involved in cAMP-mediated
dephosphorylation of pRB, we treated the cells with inhibitors of PP1 and
PP2A. The inhibitors, okadaic acid, calliculin and tautomycin, abolished the
effect of forskolin on dephosphorylation of pRB. We excluded the possibility
that this effect was due to the inhibitors activating the CDKs, as all three
phosphatase inhibitors in fact inhibited the activities of the relevant CDKs.
The results imply that cAMP may lead to activation of a pRB-directed
phosphatase, which may well explain why pRB seems to be dephosphorylated even
faster than the inhibition of the overall CDK-kinase activity, and why pRB is
partially rephosphorylated in the presence of fully active CDKs.
Although the discrepancy between the phosphorylation state of pRB and the
CDK activity could be explained by activation of a phosphatase, we can not
explain why prolonged exposure to forskolin leads to reactivation of the CDKs.
In previous reports, we observed that also levels of MYC and Mad in Reh cells
were transiently regulated by forskolin
(Blomhoff et al., 1987;
Naderi and Blomhoff, 1999
). We
showed that re-addition of forskolin every 5 hours would keep the MYC levels
permanently inhibited and the Mad levels permanently high, presumably due to
prolonged elevated levels of intracellular cAMP
(Naderi and Blomhoff, 1999
).
It is possible that re-addition of forskolin to Reh cells would lead to a
permanent reduction in the activity of the CDKs. We did not, however, test
this possibility, because the main issue in the present study was rather to
understand how cells could be permanently inhibited in G1 when pRB is
partially rephosphorylated.
To address this next question, we examined the ability of pRB to bind to
the different members of the E2F/DP family of transcription factors, after
prolonged treatment with forskolin. It is generally believed that pRB exerts
its growth inhibitory effect by binding to the members of the E2F/DP family
(Qian et al., 1992;
Qin et al., 1992
;
Ohtani, 1999
), and that pRB
binds to E2F/DP in its hypophosphorylated state
(Bagchi et al., 1991
;
Chellappan et al., 1991
;
Knudsen and Wang, 1997
;
Helin, 1998
). Our finding that
treatment of Reh cells with forskolin for 2 hours induced binding of pRB to
E2Fs was therefore in line with data from other cell systems, having shown
that growth inhibition is associated with hypophosphorylated forms of pRB
binding to E2Fs (Hatakeyama et al.,
1994
; Beijersbergen and
Bernards, 1996
; Wu et al.,
2000
). The unexpected result was, however, that the partially
rephosphorylated forms of pRB present after 72 hours of forskolin treatment
still and even more readily formed complexes with E2F-1 and E2F-4 than did the
more dephosphorylated form of pRB present after 2 hours treatment with
forskolin. We showed that the complex between pRB and E2F/DP-1 increased after
72 hours, concomitant with a reduction in free E2F/DP-1s. Also, the complexes
between cyclin A, p130 or p107 and E2F-4 increased with longer exposure to
forskolin. The functional implication of the increased complex formation
between the pocket proteins and E2Fs was confirmed by the observed inhibition
of E2F-promoter activity in vivo in the presence of high levels of cAMP. This
may reflect a cAMP-mediated repression of one or several genes important for
the G1-arrest in Reh cells. Interestingly, we can conclude that these genes
are not cyclin A or cyclin E because these genes are highly expressed, and
their associated kinase activities are fully restored at 72 hours of forskolin
treatment.
We cannot at present fully explain the mechanisms behind the increased
complex formation between partially rephosphorylated forms of the pocket
proteins and E2Fs. The most obvious explanation would be that functionally
important phosphorylation sites in the E2F-binding pocket of pRB were
permanently dephosphorylated upon prolonged exposure to forskolin. From our
results using four commercially available antibodies (Ser 780, Ser 795, Ser
807/811 and Thr 821) directed against different phosphorylation sites on pRB,
known to be in a region involved in E2F-binding
(Knudsen and Wang, 1997), we
could conclude that none of the sites were permanently dephosphorylated in the
total pRB-population. However, at least three of these sites seemed to be
dephosphorylated in a large portion of the pRB molecules, even after 72 hours
of forskolin treatment, and thus may account for the ability of pRB to still
bind E2F at this time point. These results agree with the findings that
phosphorylation on several residues in the large A/B-pocket of pRB is needed
to disrupt the E2F-pRB binding (Knudsen
and Wang, 1997
). Recent results even indicate that complete
phosphorylation of pRB is needed to relieve E2F and allow transcription from
E2F-responsive promoters (Lundberg and
Weinberg, 1998
; Brown et al.,
1999
; Harbour et al.,
1999
). Our observation of increased complex formation between pRB
and E2F at 72 hours, as compared with 2 hours of forskolin treatment, is
supported by recent reports suggesting that E2Fs more readily form complexes
with hypophosphorylated forms of pRB than with unphosphorylated forms of pRB
(Ezhevsky et al., 1997
;
Brugarolas et al., 1999
;
Ezhevsky et al., 2001
). Thus,
the fact that the most dephosphorylated forms of pRB became less apparent
after 72 hours of forskolin treatment suggest that forskolin induced a shift
in the fraction of unphosphorylated forms of pRB to more hypophosphorylated
forms.
Taken together, we have shown that forskolin-treated Reh cells are permanently arrested in G1, despite pRB becoming partially rephosphorylated upon prolonged exposure to forskolin. The growth-inhibitory potential of partially rephosphorylated pRB could be explained by inhibited E2F-promoter activity, enforced by the strong ability of rephosphorylated pRB to form complexes with E2Fs.
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Bagchi, S., Weinmann, R. and Raychaudhuri, P. (1991). The retinoblastoma protein copurifies with E2F-I, an E1A-regulated inhibitor of the transcription factor E2F. Cell 65,1063 -1072.[Medline]
Beijersbergen, R. L. and Bernards, R. (1996). Cell cycle regulation by the retinoblastoma family of growth inhibitory proteins. Biochim. Biophys. Acta 1287,103 -120.[Medline]
Beijersbergen, R. L., Kerkhoven, R. M., Zhu, L., Carlee, L., Voorhoeve, P. M. and Bernards, R. (1994). E2F-4, a new member of the E2F gene family, has oncogenic activity and associates with p107 in vivo. Genes Dev. 8,2680 -2690.[Abstract]
Bishop, J. M. (1987) The molecular genetics of cancer. Science 235,305 -311.[Medline]
Blomhoff, H. K., Smeland, E. B., Beiske, K., Blomhoff, R., Ruud, E., Bjoro, T., Pfeifer-Ohlsson, S., Watt, R., Funderud, S. and Godal, T. (1987). Cyclic AMP-mediated suppression of normal and neoplastic B cell proliferation is associated with regulation of myc and Ha-ras protooncogenes. J. Cell Physiol. 131,426 -433.[Medline]
Brown, V. D., Phillips, R. A. and Gallie, B. L.
(1999). Cumulative effect of phosphorylation of pRB on regulation
of E2F activity. Mol. Cell. Biol.
19,3246
-3256.
Brugarolas, J., Moberg, K., Boyd, S. D., Taya, Y., Jacks, T. and
Lees, J. A. (1999). Inhibition of cyclin-dependent kinase 2
by p21 is necessary for retinoblastoma protein-mediated G1 arrest after
gamma-irradiation. Proc. Natl. Acad. Sci. USA
96,1002
-1007.
Buchkovich, K., Duffy, L. A. and Harlow, E. (1989). The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell 58,1097 -1105.[Medline]
Chellappan, S. P., Hiebert, S., Mudryj, M., Horowitz, J. M. and Nevins, J. R. (1991). The E2F transcription factor is a cellular target for the RB protein. Cell 65,1053 -1061.[Medline]
Christoffersen, J., Smeland, E. B., Stokke, T., Tasken, K., Andersson, K. B. and Blomhoff, H. K. (1994). Retinoblastoma protein is rapidly dephosphorylated by elevated cyclic adenosine monophosphate levels in human B-lymphoid cells. Cancer Res. 54,2245 -2250.[Abstract]
DeCaprio, J. A., Furukawa, Y., Ajchenbaum, F., Griffin, J. D. and Livingston, D. M. (1992). The retinoblastoma-susceptibility gene product becomes phosphorylated in multiple stages during cell cycle entry and progression. Proc. Natl. Acad. Sci. USA 89,1795 -1798.[Abstract]
Durfee, T., Becherer, K., Chen, P. L., Yeh, S. H., Yang, Y., Kilburn, A. E., Lee, W. H. and Elledge, S. J. (1993). The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 7, 555-569.[Abstract]
Dyson, N. (1998). The regulation of E2F by
pRB-family proteins. Genes Dev.
12,2245
-2262.
Easton, J., Wei, T., Lahti, J. M. and Kidd, V. J. (1998). Disruption of the cyclin D/cyclin-dependent kinase/INK4/retinoblastoma protein regulatory pathway in human neuroblastoma. Cancer Res. 58,2624 -2632.[Abstract]
Ewen, M. E., Xing, Y. G., Lawrence, J. B. and Livingston, D. M. (1991). Molecular cloning, chromosomal mapping, and expression of the cDNA for p107, a retinoblastoma gene product-related protein. Cell 66,1155 -1164.[Medline]
Ezhevsky, S. A., Nagahara, H., Vocero-Akbani, A. M., Gius, D.
R., Wei, M. C. and Dowdy, S. F. (1997). Hypo-phosphorylation
of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in
active pRb. Proc. Natl. Acad. Sci. USA
94,10699
-10704.
Ezhevsky, S. A., Ho, A., Becker-Hapak, M., Davis, P. K. and
Dowdy, S. F. (2001). Differential regulation of
retinoblastoma tumor suppressor protein by g(1) cyclin-dependent kinase
complexes in vivo. Mol. Cell. Biol.
21,4773
-4784.
Ginsberg, D., Vairo, G., Chittenden, T., Xiao, Z. X., Xu, G., Wydner, K. L., DeCaprio, J. A., Lawrence, J. B. and Livingston, D. M. (1994). E2F-4, a new member of the E2F transcription factor family, interacts with p107. Genes Dev. 8,2665 -2679.[Abstract]
Goodrich, D. W., Wang, N. P., Qian, Y. W., Lee, E. Y. and Lee, W. H. (1991). The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle. Cell 67,293 -302.[Medline]
Hannon, G. J., Demetrick, D. and Beach, D. (1993). Isolation of the Rb-related p130 through its interaction with CDK2 and cyclins. Genes Dev. 7,2378 -2391.[Abstract]
Harbour, J. W. and Dean, D. C. (2000). The
Rb/E2F pathway: expanding roles and emerging paradigms. Genes
Dev. 14,2393
-2409.
Harbour, J. W., Luo, R. X., Dei, S. A., Postigo, A. A. and Dean, D. C. (1999). Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1. Cell 98,859 -869.[Medline]
Hatakeyama, M., Brill, J. A., Fink, G. R. and Weinberg, R. A. (1994). Collaboration of G1 cyclins in the functional inactivation of the retinoblastoma protein. Genes Dev. 8,1759 -1771.[Abstract]
Helin, K. (1998). Regulation of cell proliferation by the E2F transcription factors. Curr. Opin. Genet. Dev. 8,28 -35.[Medline]
Johnson, D. G., Schwarz, J. K., Cress, W. D. and Nevins, J. R. (1993). Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature 365,349 -352.[Medline]
Kawamata, S., Sakaida, H., Hori, T., Maeda, M. and Uchiyama,
T. (1998). The upregulation of p27Kip1 by rapamycin results
in G1 arrest in exponentially growing T-cell lines.
Blood 91,561
-569.
Knudsen, E. S. and Wang, J. Y. (1997). Dual mechanisms for the inhibition of E2F binding to RB by cyclin-dependent kinase-mediated RB phosphorylation. Mol. Cell. Biol. 17,5771 -5783.[Abstract]
Lam, E. W. and Watson, R. J. (1993). An E2F-binding site mediates cell-cycle regulated repression of mouse B- myb transcription. EMBO J. 12,2705 -2713.[Abstract]
Li, J. M., Hu, P. P., Shen, X., Yu, Y. and Wang, X. F.
(1997). E2F4-RB and E2F4-p107 complexes suppress gene expression
by transforming growth factor beta through E2F binding sites. Proc.
Natl. Acad. Sci. USA 94,4948
-4953.
Liu, C. W., Wang, R. H., Dohadwala, M., Schonthal, A. H.,
Villa-Moruzzi, E. and Berndt, N. (1999). Inhibitory
phosphorylation of PP1alpha catalytic subunit during the G(1)/S transition.
J. Biol. Chem. 274,29470
-29475.
Lundberg, A. S. and Weinberg, R. A. (1998).
Functional inactivation of the retinoblastoma protein requires sequential
modification by at least two distinct cyclin-cdk complexes. Mol.
Cell. Biol. 18,753
-761.
Maelandsmo, G. M., Florenes, V. A., Hovig, E., Oyjord, T., Engebraaten, O., Holm, R., Borresen, A. L. and Fodstad, O. (1996). Involvement of the pRb/p16/cdk4/cyclin D1 pathway in the tumorigenesis of sporadic malignant melanomas. Br. J. Cancer 73,909 -916.[Medline]
Mayol, X., Grana, X., Baldi, A., Sang, N., Hu, Q. and Giordano, A. (1993). Cloning of a new member of the retinoblastoma gene family (pRb2) which binds to the E1A transforming domain. Oncogene 8,2561 -2566.[Medline]
Miyatake, S., Nakano, H., Park, S. Y., Yamazaki, T., Takase, K., Matsushime, H., Kato, A. and Saito, T. (1995). Induction of G1 arrest by down-regulation of cyclin D3 in T cell hybridomas. J. Exp. Med. 182,401 -408.[Abstract]
Naderi, S. and Blomhoff, H. K. (1999). Mad1 expression in the absence of differentiation: effect of cAMP on the B-lymphoid cell line Reh. J. Cell Physiol. 178, 76-84.[Medline]
Naderi, S., Gutzkow, K. B., Christoffersen, J., Smeland, E. B. and Blomhoff, H. K. (2000). cAMP-mediated growth inhibition of lymphoid cells in G1: rapid down- regulation of cyclin D3 at the level of translation. Eur. J. Immunol. 30,1757 -1768.[Medline]
Nelson, D. A. and Ludlow, J. W. (1997). Characterization of the mitotic phase pRb-directed protein phosphatase activity. Oncogene 14,2407 -2415.[Medline]
Ohtani, K. (1999). Implication of transcription factor E2F in regulation of DNA replication. Front. Biosci. 4,D793 -D804.[Medline]
Pagano, M., Pepperkok, R., Verde, F., Ansorge, W. and Draetta, G. (1992). Cyclin A is required at two points in the human cell cycle. EMBO J. 11,961 -971.[Abstract]
Pardee, A. B. (1974). A restriction point for control of normal animal cell proliferation. Proc. Natl. Acad. Sci. USA 71,1286 -1290.[Abstract]
Pardee, A. B. (1989). G1 events and regulation of cell proliferation. Science 246,603 -608.[Medline]
Pokrovskaja, K., Ehlin-Henriksson, B., Bartkova, J., Bartek, J., Scuderi, R., Szekely, L., Wiman, K. G. and Klein, G. (1996) Phenotype-related differences in the expression of D-type cyclins in human B cell-derived lines. Cell Growth Differ. 7,1723 -1732.[Abstract]
Qian, Y., Luckey, C., Horton, L., Esser, M. and Templeton, D. J. (1992). Biological function of the retinoblastoma protein requires distinct domains for hyperphosphorylation and transcription factor binding. Mol. Cell. Biol. 12,5363 -5372.[Abstract]
Qin, X. Q., Chittenden, T., Livingston, D. M. and Kaelin, W. G. J. (1992). Identification of a growth suppression domain within the retinoblastoma gene product. Genes Dev. 6, 953-964.[Abstract]
Rosenfeld, C., Goutner, A., Choquet, C., Venuat, A. M., Kayibanda, B., Pico, J. L. and Greaves, M. F. (1977). Phenotypic characterisation of a unique non-T, non-B acute lymphoblastic leukaemia cell line. Nature 267,841 -843.[Medline]
Sellers, W. R. and Kaelin, W. G., Jr (1997). Role of the retinoblastoma protein in the pathogenesis of human cancer. J. Clin. Oncol. 15,3301 -3312.[Abstract]
Sellers, W. R., Rodgers, J. W. and Kaelin, W. G. J. (1995). A potent transrepression domain in the retinoblastoma protein induces a cell cycle arrest when bound to E2F sites. Proc. Natl. Acad. Sci. USA 92,11544 -11548.[Abstract]
Sherr, C. J. (1996). Cancer cell cycles.
Science 274,1672
-1677.
Takahashi, R., Hashimoto, T., Xu, H. J., Hu, S. X., Matsui, T., Miki, T., Bigo-Marshall, H., Aaronson, S. A. and Benedict, W. F. (1991). The retinoblastoma gene functions as a growth and tumor suppressor in human bladder carcinoma cells. Proc. Natl. Acad. Sci. USA 88,5257 -5261.[Abstract]
Tan, X., Martin, S. J., Green, D. R. and Wang, J. Y.
(1997). Degradation of retinoblastoma protein in tumor necrosis
factor- and CD95-induced cell death. J. Biol. Chem.
272,9613
-9616.
Vairo, G., Livingston, D. M. and Ginsberg, D. (1995). Functional interaction between E2F-4 and p130: evidence for distinct mechanisms underlying growth suppression by different retinoblastoma protein family members. Genes Dev. 9, 869-881.[Abstract]
Weinberg, R. A. (1995). The retinoblastoma protein and cell cycle control. Cell 81,323 -330.[Medline]
Weintraub, S. J., Prater, C. A. and Dean, D. C. (1992). Retinoblastoma protein switches the E2F site from positive to negative element. Nature 358,259 -261.[Medline]
Wu, L., Goodwin, E. C., Naeger, L. K., Vigo, E., Galaktionov,
K., Helin, K. and DiMaio, D. (2000). E2F-Rb complexes
assemble and inhibit cdc25A transcription in cervical carcinoma cells
following repression of human papillomavirus oncogene expression.
Mol. Cell. Biol. 20,7059
-7067.