The retinoblastoma susceptibility gene, Rb-1, was
cloned on the basis of its biallelic inactivation in human
retinoblastomas(1, 2) . Rb-1 was subsequently
observed to be mutated in a variety of tumor types, indicating that it
may play a general role in the inhibition of the transformed
phenotype(3, 4) . Reintroduction of Rb-1 into
some Rb-/- cells can lead to decreased
tumorigenicity in nude mice or inhibition of growth in
culture(5) . Furthermore, ectopic expression of Rb-1
or microinjection of RB protein can block cell cycle progression at
G
/S(6, 7, 8) .
RB forms
complexes with many proteins, and this protein binding activity is
required for growth suppression. RB binds to its target proteins by
several different mechanisms(1) . Viral oncoproteins such as
the SV40 large T-antigen (T-Ag), (
)and several cellular
proteins, e.g. D-type cyclins and Elf-1, contain the
LXCXE motif that is important for binding to the A/B
pocket of RB(9, 10, 11) . The E2F
transcription factors do not contain the LXCXE motif
and their binding requires the A/B pocket and C-terminal amino acids,
and this E2F binding site is called the ``large A/B
pocket''(7, 12, 13, 14) . The
C-terminal region of RB also contains an A/B pocket-independent binding
domain, the C pocket, which binds to the nuclear c-Abl tyrosine kinase (15) . The large A/B pocket and the C pocket of RB do not
overlap, because RB can simultaneously bind to E2F and c-Abl in
vitro and in vivo(16) . In addition, complexes
containing T-Ag/RB/c-Abl and cyclin D2/RB/c-Abl have been
detected(15, 16) . The protein assembly function of RB
is known to be required for growth suppression, since overexpression of
the individual domains can inactivate RB biological
function(16) .
The protein binding function of RB is
regulated by phosphorylation(1) . RB phosphorylation is
observed as cells progress from G
into S phase of the cell
cycle, and this is correlated with the disruption of RB-assembled
protein complexes. RB contains 16 Ser/Thr-Pro motifs which are
potential Cdk phosphorylation sites. At least seven of these sites
(Ser-249, -807, -811, and Thr-252, -373, -821, and -826) have been
shown to be phosphorylated in vivo(17, 18, 19) . A number of other sites
are also phosphorylated in vivo, but the exact identity of
these sites is unknown(17, 18, 19) . In
mitotic cells, cdc2/cyclin B is the principle RB kinase(19) .
In interphase cells, RB is phosphorylated by other Cdk-cyclin
complexes, including Cdk4/cyclin D and Cdk2/cyclin A(2) .
Several Cdk consensus phosphorylation sites have been mutated in human
and murine RB(9, 20, 21, 22) .
Elimination of eight Cdk consensus phosphorylation sites in murine RB
abolishes phosphorylation in vivo, and correlates with a more
effective suppression of E2F or Elf-1 activity in transfected
cells(11, 22) . The previous analysis of
phosphorylation site mutants of RB did not address the question of
whether the multiple Cdk phosphorylation sites of RB had redundant
regulatory function.
Since RB can bind its target proteins through
at least three different mechanisms, we hypothesized that each protein
binding function might be regulated by distinct phosphorylation
sites(1) . This hypothesis would suggest that the multiple
phosphorylation sites can establish a collection of functional states
of RB, depending on the specific sites phosphorylated. To address this
hypothesis, we developed in vitro binding assays to examine
the requirement of specific phosphorylation sites on RB protein binding
activity. We show here that different phosphorylation sites are
required to inhibit the binding to T-Ag, c-Abl, and E2F. Furthermore,
specific sites are also required for the efficient phosphorylation of
RB in vivo and the reversal of RB-mediated growth suppression
by cyclin A.
MATERIALS AND METHODS
Cell Culture
C33-A, HeLa, SAOS-2, and COS cells
were obtained from the American Type Culture Collection. C33-A and
SAOS-2 cells were cultured in Dulbecco's modified Eagle's
medium supplemented with 10 and 15% fetal calf serum, respectively, at
37° C. COS and HeLa cells were cultured in Dulbecco's
modified Eagle's medium supplemented with 10% calf serum at
37° C. Sf9 cells were cultured at 25° C in Grace's medium
containing 10% heat inactivated fetal calf serum, yeastolate,
lactalbumin hydrolysate, and gentamicin.
Plasmids
The RB phosphorylation site mutants
PSM.2S and PSM.4 were constructed by oligonucleatide-directed
mutagenesis using the Mutagene in vitro mutagenesis kit
(Bio-Rad). PSM.2S was constructed with the primer
CTATATTGCACCCCTGAAGCTTCC. PSM.4 was constructed by further mutagenizing
PSM.2S using the primer GGTCTGCCGGCACCAACAAAAATGGCTCCAAG. PSM.2T was
constructed by polymerase chain reaction subcloning the T821A/T826A
mutation from PSM.4 into WT-RB. The WT GST-RB constructs Ase-End,
Ssp-End, and Mun-End have been previously described(15) . The
PSM.2S, PSM.2T, and PSM.4 GST-RB fusions were constructed by replacing
restriction fragments in WT-RB GST fusion constructs with those from
each of the PSM clones. The pCMV-FL: WT, PSM.2S, PSM.2T, and PSM.4
expression plasmids, containing the full-length RB cDNA were
constructed by cloning into the unique BamHI site of
pCMV-Neo(23) . The CMV-CycA(8) , pBABE-Puro (24) , RSV-T-Ag(25) , and GST-E2F-1 (13) plasmids have been previously described. Recombinant SV40
large T-Ag baculovirus was described in Melendy and
Stillman(26) .
Transfections
The BES-calcium phosphate method was
used to transfect all cells(27) . Cells were washed three times
with phosphate-buffered saline 14-20 h after the addition of DNA.
In transient assays, cells were harvested approximately 48 h after
washing. All transfections were carried out using 16 µg of total
DNA/100-mm dish. For the expression of FL-RB in C33-A cells, 10 µg
of RC-CMV-CycA and 6 µg of the CMV-RB constructs were transfected.
For co-immunoprecipitation of RB with T-Ag we used 8 µg of
CMV-CycA, 4 µg of RSV-T-Ag, and 4 µg of CMV-RB. For COS
transfections 16 µg of CMV-RB alone were transfected. For SAOS-2
transfections, 9 µg of effector (either RC-CMV-CycA or RSV-T-Ag), 6
µg of CMV-RB, and 1 µg of pBABE-Puro was used. For the flat
cell formation assays, transfected SAOS-2 cells were split into
Dulbecco's modified Eagle's medium supplemented with 15%
fetal calf serum and puromycin at 0.5 mg/ml approximately 24 h after
washing. Selection was carried out for 7-9 days, plates were
stained with crystal violet, and the number of flat cells on the plate
was determined by counting random fields as described
previously(8) .
Immunoprecipitation and Immunoblotting
In vivo phosphate labeling of RB was carried out essentially as described
previously(20) . For the immunoprecipitation of
phosphate-labeled RB, transfected cells were harvested by scraping and
lysed in RIPA buffer (25 mM Tris, pH 7.5, 50 mM NaCl,
0.5% sodium deoxycholate, 0.1% SDS, and 0.2% Nonidet P-40) supplemented
with protease inhibitors (10 µg/ml 1,10-phenanthroline, 10
µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride), and phosphatase inhibitors (10
mM sodium fluoride, 10 mM sodium phosphate, 10 mM sodium pyrophosphate). Lysates were clarified by centrifugation at
15,000
g for 10 min at 4° C followed by filtration
through a 0.45-µm syringe filter (Nalgene). Clarified lysates were
then pre-cleared by incubation with 9 µg of rabbit anti-mouse
antibody (Cappel) and 15 µl of protein A-Sepharose (Pharmacia) at
4° C for 30 min, followed by centrifugation at 2000
g, then subjected to specific immunoprecipitation. For
immunoprecipitation from unlabeled cells, transfected cells were
harvested by scraping and lysed in NNT-N buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 0.5% Nonidet P-40, 1
mM EDTA). Lysates were clarified by centrifugation at 15,000
g for 10 min at 4° C. Proteins were
immunoprecipitated from the clarified lysates using the following
antibodies: for RB, 1.5 µg of G3-245 monoclonal antibody
(Pharmingen) and for T-Ag, 1.5 µg of pAB 114 monoclonal antibody
(Pharmingen). Immunoprecipitations were carried out for 4 h at 4° C
using 4.5 µg of rabbit anti-mouse and protein A-Sepharose in a
volume of 0.5-1.0 ml. Immunocomplexes were recovered by
centrifugation at 2000
g, and washed 4-8 times
with 1.0 ml of NNT-N or RIPA buffer. Immunoprecipitated proteins were
separated by SDS-PAGE and transferred to Immobilon-P membrane
(Millipore).Immunoblotting was carried out with the following
antibodies: full-length RB proteins were detected with the G3-245
monoclonal antibody (Pharmingen), GST-RB proteins were detected with
either 851 serum (15) or the XZ91 monoclonal antibody
(Pharmingen), and T-Ag was detected with the pAB 114 monoclonal
antibody (Pharmingen). Immunoblots were developed using either alkaline
phosphatase-conjugated secondary antibody for colorimetric development
(Fisher), or horseradish peroxidase-conjugated secondary antibodies
(Cappel, Life Technologies, Inc.) for enhanced chemiluminescent (ECL)
development (Amersham).
In Vitro Kinase Reactions
Active Cdk/cyclin
kinases used to phosphorylate RB were obtained from two sources.
Cdk2/cycA was purified from recombinant baculovirus-infected Sf9 cells
(J. W. Harper). Alternatively, active cdc2 was immunopurified from
nocodazole-arrested mitotic HeLa cells as described
before(19) . Kinase reactions with either active cdc2 (immune
complexes from 5 to 10
10
mitotic cells) or
purified Cdk2/cycA (final concentration 1-5 nM) were
carried out in 50 µl of kinase buffer using 150 µM ATP, 80-200 µCi of [
-
P]ATP,
and 10-30 ng of RB as substrate. Reactions were allowed to
proceed for 30-45 min with shaking at room temperature. The
resulting phosphorylated protein was then either resolved by SDS-PAGE,
transferred to Immobilon-P, and detected by autoradiography and
immunoblotting, used in the determination of stoichiometry of
phosphorylation (see below), or utilized in in vitro binding
assays (see below).
Quantitation
For the determination of the
stoichiometry of phosphorylation with in vitro phosphorylated
proteins, the phosphorylated
P-labeled proteins were first
resolved by SDS-PAGE. The incorporated phosphate was then determined by
liquid scintillation counting of the labeled bands, from which the
phosphate molecules per protein molecule were determined. For the
determination of the relative phosphorylation in vivo,
immunoprecipitated metabolically labeled proteins were resolved by
SDS-PAGE and detected by autoradiography, followed by immunoblotting.
The autoradiograms (
P) and immunoblots (protein) were
quantitated with an LKB densitometer, and the
P/protein
ratio was normalized to WT, which was arbitrarily set to 100.
Binding Assays
GST fusion proteins were expressed
in bacteria and purified on glutathione-agarose as described previously (15) . The binding of RB to c-Abl was also carried out as
described previously(15) . For binding of soluble GST-AE to
immobilized T-Ag, 1.0 µg of T-Ag was immobilized by
immunoprecipitation with pAB 114. Approximately 200 ng of soluble
GST-AE protein, obtained by elution with glutathione in 0.5 ml of
NNT-N, was added to immobilized T-Ag and incubated for 35-45 min
at 4° C, then washed 4 times with NNT-N. Bound protein was
solubilized by boiling in SDS buffer and resolved by 7.5% SDS-PAGE. The
GST-RB proteins were then detected by autoradiography and immunoblot.
For the binding of soluble T-Ag to GST-AE, approximately 30 ng of each
of the GST-AE fusions (WT, PSM.2S, PSM.2T, and PSM.4) were immobilized
on glutathione-agarose. Sf9 cells infected with recombinant T-Ag
baculovirus were lysed in NNT-N supplemented with phosphatase and
protease inhibitors, and clarified by centrifugation. Approximately 500
ng of T-Ag in 0.5 ml of NNT-N were added to the immobilized GST-AE and
incubated at 4° C for 45 min. The GST-AE beads were then washed
five times with NNT-N. The bound protein was solubilized by boiling in
SDS buffer and resolved by 8% SDS-PAGE, and T-Ag was visualized by
immunoblot. The E2F supershift assay was carried out using 30 ng of
soluble of GST-AE and E2F purified from HeLa cells as described
previously(16) .RB produced in transfected C33-A cells was
utilized in binding to immobilized, GST-c-Abl, GST-E2F-1, T-Ag, GST-E7,
and GST-Elf1. The binding of RB to GST-Abl was carried out as described
previously(15) . For binding to T-Ag, GST-E7, or GST-Elf1,
cells were lysed with NNT-N (including protease and phosphatase
inhibitors), whereas for binding to GST-E2F-1, cells were lysed with
NNT-0.1% N (NNT-N with 0.1% Nonidet P-40). Lysates were clarified by
centrifugation, and then incubated with immobilized T-Ag (200-300
ng), GST-Elf1 (1-1.5 µg), or GST-E2F-1 (500-700 ng) for
1 h at 4° C. The bound protein was recovered by centrifugation at
2000
g and washed 3-4 times with the lysis
buffer.
RESULTS
Construction and Characterization of RB Phosphorylation
Site Mutants
We focused our analysis on four Cdk consensus
phosphorylation sites in RB, Ser-807, Ser-811, Thr-821, and Thr-826.
These four sites were chosen because they are phosphorylated to a
higher level than other sites in HeLa and Molt4 cells, suggesting that
they are phosphorylation sites of physiological relevance(19) .
These four sites can also be phosphorylated in vitro by
cdc2/cyclin B or Cdk2/cyclin A(19) . We constructed double
mutants of Ser-807/811 or Thr-821/826, because we were intrigued by the
close proximity of the two Ser and the two Thr consensus sites. Both
Ser-807 and -811 were mutated in Phosphorylation Site Mutant 2S (PSM.2S, Fig. 1). In PSM.2T, Thr-821 and -826
were mutated (Fig. 1). The PSM.4 combines the mutations in
PSM.2S and PSM.2T. The WT and PSM proteins were expressed in bacteria
as GST fusions in two forms: the GST-SE, which only contained the C
pocket; or the GST-AE, which contained the A/B and C pockets of RB (Fig. 1). They were also expressed in mammalian cells as
full-length RB proteins.
Figure 1:
Summary of RB constructs. RB
phosphorylation site mutants (PSM) were created by
oligonucleotide-directed mutagenesis, as described under
``Materials and Methods.'' RB contains 16 S/T-P sites (Thr-5,
Ser-230, Ser-249, Thr-252, Thr-356, Thr-373, Ser-567, Ser-608, Ser-612,
Ser-780, Ser-788, Ser-795, Ser-807, Ser-811, Thr-821, Thr-826) which
are denoted by arrows (the filled arrows depict
serine while the open arrows threonine sites). PSM.2S is a
double substitution of Ser-807 and -811 with Ala and Leu, respectively.
PSM.2T is a double mutant of Thr-821 and -826 substituted to Ala. PSM.4
is the combination of PSM.2S and PSM.2T. C706F is an A/B pocket mutant
of RB. The RB proteins used for in vitro binding experiments
were expressed in bacteria as glutathione S-transferase (GST)
fusion proteins. The GST-AE fusion contains RB amino acids
384-928. WT AE contains 10 S/T-P sites and functional A/B and C
pockets. The GST-SE contains RB amino acids 768-928. The SE
fragment of RB contains seven S/T-P sites and a functional C pocket.
The GST-ME fusion protein containing RB amino acids 835-928, has
no C-pocket activity and no S/T-P sites.
Stoichiometric Phosphorylation of RB in Vitro
The in vitro phosphorylation of WT RB with Cdk/cyclin could
inhibit its protein binding activity as shown below. However, the mere
incorporation of phosphate into RB did not necessarily cause an
inhibition of binding, because some of the
P-labeled WT RB
was found to bind to its target proteins (data not shown). This result
suggested that some of the phosphorylation sites might be irrelevant
for the inhibition of protein binding. Therefore, we developed
conditions to achieve the quantitative phosphorylation of RB in
vitro at all Cdk consensus sites, using mitotic cdc2 kinase or
purified Cdk2/cycA. Quantitative phosphorylation was indicated by the
complete conversion of RB to a single band with slower electrophoretic
mobility on SDS-PAGE (Fig. 2, compare lanes 1 and 2). The stoichiometry of phosphorylation was measured for WT
and PSM RB, and found to be in agreement with the number of Cdk
consensus phosphorylation sites present in each protein (Table 1A). GST is not phosphorylated by Cdk/cyclin(18) .
Along with the lower stoichiometry, the phosphorylated PSM proteins
also exhibited a reduced shift in their electrophoretic mobility. The
phosphorylated WT RB migrated to a higher position than its
corresponding phosphorylation site mutants (Fig. 2, compare lanes 2-5 with lanes 6-8). Thus, the
stoichiometry of phosphorylation appears to dictate the degree of
mobility shift.
Figure 2:
Phosphorylation of PSM RB in vitro. Purified GST-AE: WT (lanes 1 and 2) PSM.2S (lane 3), PSM.2T (lane 4), PSM.4 (lane 5);
or SE: WT (lane 6), PSM.2S (lane 7), PSM.4 (lane
8), or ME (lane 9) were phosphorylated with Cdk2/cyclin A (lanes 1-5) or cdc2 immunoprecipitated from mitotic HeLa
cells (lanes 6-9). Kinase reactions were carried out in
the absence (lane 1) or presence (lanes 2-9) of
150 µM ATP and 80 µCi of
[
-
P]ATP. The GST-AE proteins were resolved
on 8% SDS-PAGE (lanes 1-5). The SE proteins were
resolved on 15% SDS-PAGE (lanes 6-9). Proteins were
transferred to Immobilon-P, and detected by autoradiography (
P, upper panels) followed by anti-RB
immunoblotting (
RB, lower
panels)
In Vitro Binding Assays
The quantitatively
phosphorylated WT and PSM RB were used in two in vitro assays
to examine the regulation of protein binding activity. In the first
assay (Fig. 3), GST-RB proteins were labeled with
P. Each labeled sample was combined with a known amount of
its unphosphorylated counterpart to give a constant
P/protein ratio, and then applied to immobilized c-Abl or
T-Ag. The
P/protein ratio of the input and the bound
fraction was compared for each sample. If phosphorylation inhibited
binding, the
P/protein ratio of the bound fraction would
be lower than that of the input. However, if phosphorylation did not
affect binding, the
P/protein ratio of the bound fraction
would be equal to that of the input. In the second assay (Fig. 4), GST-RB proteins were quantitatively phosphorylated.
The RB proteins were then used either in an immobilized form to bind
soluble c-Abl or T-Ag (Fig. 4, B and C), or in
a soluble form to interact with E2F in gel shift assays (Fig. 4D).
Figure 3:
In vitro phosphorylation inhibits
the protein binding function of RB. A, phosphorylation
inhibits binding to c-Abl. RB-SE or ME fragments (lanes
1-4) obtained by thrombin cleavage were quantitively
phosphorylated with mitotic cdc2 kinase. Each phosphorylated protein
was combined with a 10-fold excess of its unphosphorylated counterpart,
and this was utilized as the Input. Each sample was assayed for binding
to GST-A1 (the ATP-binding lobe of c-Abl fused with GST(15) ).
The Input (upper panels) represent 10% of the sample applied
to the binding reaction. Protein fractions were resolved by 15%
SDS-PAGE and transferred to Immobilon-P. Phosphorylated SE fragments
were visualized by autoradiography (
P, left
panels), and the unphosphorylated SE or ME were revealed by
anti-RB immunoblot (
RB, right panels). B,
phosphorylation inhibits binding to T-Ag. Soluble GST-AE proteins were
quantitatively phosphorylated with mitotic cdc2 kinase. Each
phosphorylated protein was combined with a 25-fold excess of its
unphosphorylated counterpart, and this was assayed for binding to SV40
large T-antigen immobilized by immunoprecipitation with protein
A-Sepharose. The input and the bound fraction of WT (lane 1),
PSM.2S (lane 2), PSM.4 (lane 3), and C706F (lane
4) were analyzed by autoradiography (
P, left
panels) to detect phosphorylated RB and anti-RB immunoblot
(
RB, right panels) to detect the excess unphosphorylated
GST-AE. 15% of the input and all of the bound fraction was loaded.
Samples were resolved by 7.5% SDS-PAGE.
Figure 4:
Differential protein binding activities of in vitro phosphorylated RB. A, GST-AE fragments used
in binding assays. GST-AE proteins WT (lanes 1 and 5), PSM.2S (lanes 2 and 6), PSM.2T (lanes 3 and 7), and PSM.4 (lanes 4 and 8), were immobilized on GSH-agarose and incubated with
Cdk2/cycA in the absence (lanes 1-4) or presence (lanes 5-8) of 150 µM ATP. A portion of
each sample was resolved by 8% SDS-PAGE, transferred to Immobilon-P,
then immunoblotted with anti-RB antibody. B, phosphorylation
of Ser-807/811 is required to inhibit c-Abl binding. Equal amounts of
GST-AE proteins, either unphosphorylated (lanes 1-4) or
phosphorylated (lanes 5-9) as shown in A, were
incubated with in vitro translated c-Abl, labeled with
[
S]methionine. The c-Abl bound to immobilized
GST-AE, WT (lanes 1 and 5), PSM.2S (lanes 2 and 6), PSM.2T (lanes 3 and 7), or
PSM.4 (lanes 4 and 8), was recovered and resolved by
6.5% SDS-PAGE. The amount of c-Abl bound was determined by
autofluorography. C, phosphorylation of Thr-821/826 is
required to inhibit T-Ag. binding. The immobilized GST-AE proteins,
either unphosphorylated (lanes 1-4) or phosphorylated (lanes 5-8) as shown in A, were incubated with
lysate of Sf9 cells infected with a recombinant baculovirus expressing
T-Ag. The T-Ag bound to GST-AE, WT (lanes 1 and 5),
PSM.2S (lanes 2 and 6), PSM.2T (lanes 3 and 7), or PSM.4 (lanes 4 and 8), was resolved
by 8% SDS-PAGE and transferred to Immobilon-P. The amount of bound T-Ag
was determined by immunoblotting with antibodies for T-Ag. D,
phosphorylation at Ser-807/811 and Thr-821/826 is dispensible for
inhibiting binding to E2F. The unphosphorylated GST-AE proteins (lanes 1-4 and 9), the phosphorylated GST-AE
proteins (lanes 5-8), or the GST-SE protein (lane 10) were purified and added to DNA binding reactions, as
described under ``Materials and Methods.'' To prevent the
phosphorylation of E2F by Cdk2/cyclin A, excess EDTA was added to the
GST-AE preparations after phosphorylation but prior to incubation with
E2F. The GST-SE protein, lacking the A/B pocket, does not cause any
shift in the electrophoretic mobility of the E2F
DNA complex (lane 10). Unphosphorylated GST-AE, WT (lanes 1 and 9), PSM.2S (lane 2), PSM.2T (lane 3), and
PSM.4 (lane 4), all formed complexes with E2F, shifting the
mobility of the E2F
DNA complex to a position labeled E2F/RB.
Phosphorylated GST-AE, WT (lane 5), PSM.2S (lane 6),
PSM.2T (lane 7), and PSM.4 (lane 8), all failed to
complex with E2F, having no effect on the mobility of the E2F
DNA
complex. The addition of Cdk2/cycA under these experimental conditions
had no observable effect on the E2F
DNA complex (compare lanes
1 and 8).
Ser-807/811 Are Required to Inhibit c-Abl
Binding
Quantitative phosphorylation of the WT SE fragment of RB
could be shown to inhibit c-Abl binding in vitro (Fig. 3A, lane 1). This inhibition was indicated
by a 15-20-fold reduction in the
P/protein ratio of
the bound fraction (Fig. 3A, compare lane 1 of
the two panels). Unlike WT SE, the phosphorylated PSM.2S could bind to
c-Abl, and the
P/protein ratios of the input and bound
fractions were similar (compare lane 2 of the two panels).
PSM.4 behaved in a fashion identical to PSM.2S (Fig. 3A,
lane 3). The ME fragment of RB did not bind to c-Abl,
demonstrating specificity in the binding reaction (lane 4).
This result suggested that phosphorylation at Ser-807/811 was necessary
for the inhibition of RB/c-Abl interaction.The same result was
obtained with the second in vitro assay (Fig. 4B). Equal amounts of unphosphorylated (Fig. 4A, lanes 1-4) and phosphorylated WT and
PSM GST-AE (Fig. 4A, lanes 5-8) were used in the
binding reactions. The unphosphorylated WT or PSM proteins bound the
same amount of c-Abl, which was translated in vitro and
labeled with [
S]methionine (Fig. 4B,
lanes 1-4). Phosphorylation of WT RB reduced c-Abl binding
to background level (compare lanes 1 and 5).
Phosphorylation of PSM.2S and PSM.4, again, had no effect on RB/c-Abl
complex formation (Fig. 4B, compare lanes 2 and 4 with 6 and 8). Phosphorylation of
PSM.2T did cause an inhibition of c-Abl binding (Fig. 4B,
lanes 3 and 7). Taken together, these results show that
phosphorylation of Ser-807/811 is required to inhibit the C pocket
function of RB, while the phosphorylation of Thr-821/826 is not
required. In the absence of Ser-807/811, phosphorylation of the
remaining eight Cdk consensus sites in the AE fragment of RB cannot
disrupt the RB/c-Abl interaction.
Thr-821/826 Are Required to Inhibit T-Ag
Binding
The inhibition of T-Ag binding by phosphorylation could
also be demonstrated in vitro, as shown by a 10-15-fold
reduction in the
P/protein ratio of the WT GST-AE fraction
bound to immobilized T-Ag (Fig. 3B, lane 1). The RB
mutant C706F failed to bind to T-Ag under the experimental conditions (lane 4), showing specificity for the binding reactions. The
phosphorylated PSM.2S showed a reduced affinity for T-Ag, comparable to
that of the WT (Fig. 3B, compare lanes 1 and 2). In contrast, phosphorylated PSM.4 was capable of binding
to T-Ag, as indicated by the similar
P/protein ratio in
the input and the bound fractions (Fig. 3B, lane 3).
The same result was again obtained with the second in vitro assay. The unphosphorylated WT or PSM proteins bound equal amounts
of T-Ag (Fig. 4C, lanes 1-4). Phosphorylation of
WT and PSM.2S led to an inhibition of T-Ag binding (Fig. 4C,
lanes 5 and 6). However, the phosphorylated PSM.2T
protein retained full activity to complex with T-Ag (Fig. 4C, compare lanes 3 and 7). As
expected, PSM.4 behaved like PSM.2T (Fig. 4C, compare lane 4 with 8). These results showed that
phosphorylation at Thr-821/826, but not at Ser-807/811, is necessary
for the inhibition of T-Ag binding.
Ser-807/811 and Thr-821/826 Are Not Required to Inhibit
E2F Binding
To assay the E2F binding activity, the
unphosphorylated or phosphorylated GST-AE fragments were added to
purified E2F and an E2F-oligonucleotide (Fig. 4D). In
the unphosphorylated form, the WT and the PSM fragments tested all
bound to E2F, as indicated by the ``supershift'' of the
E2F
DNA complex (lanes 1-4). All four proteins
failed to complex with E2F
DNA when phosphorylated (lanes
5-8). As a negative control, the GST-SE fragment of RB did
not supershift the E2F
DNA complex (lane 10). These
results showed that Cdk sites within the AE fragment of RB were
sufficient to inhibit E2F binding. However, phosphorylation of
Ser-807/811 and Thr-821/826 are not required to inhibit E2F binding.
In Vivo Phosphorylation of PSM RB
With the in
vitro quantitatively phosphorylated RB, we showed that different
Cdk sites were required to regulate binding to c-Abl, T-Ag, and E2F.
Because not all of the Cdk sites were quantitatively phosphorylated in vivo, we wished to determine the protein binding activity
of in vivo phosphorylated WT and PSM RB. The WT and PSM
proteins were transiently expressed in the human cervical carcinoma
cell line, C33-A, which contains a truncated RB that is unstable.
Phosphorylation of the exogeneously expressed RB was enhanced by the
cotransfection with cyclin A. The relative extent of phosphorylation
was determined by the appearance of slower migrating bands on anti-RB
immunoblots and by
P labeling (Fig. 5). The WT and
PSM.2T were phosphorylated in vivo, as indicated by the slower
migrating bands which were
P-labeled (Fig. 5, lanes 1 and 3, upper and lower panels). The
PSM.2S and PSM.4 proteins were labeled with
P in vivo (Fig. 5, lanes 2 and 4, upper panel), but
did not generate the characteristic mobility shift (Fig. 5, lanes 2 and 4, lower panel). The relative level of in vivo phosphorylation was determined for each protein (Table 1B). PSM.2T was phosphorylated to 80-90% that of WT,
consistent with the mutation of two out of 16 phosphorylation sites (Table 1B). The relative phosphorylation for PSM.2S or PSM.4 was
only about 40% that of WT (Table 1B). The inefficient
phosphorylation of the Ser-807/811 mutant RB was consistent with a
previous report which showed that mutation of the murine equivalent of
Ser-807/811 prevented the hyperphosphorylation of murine RB in
vivo(21) .
Figure 5:
Phosphorylation of PSM RB in vivo. C33-A cells were cotransfected with plasmids expressing cyclin A
and the full-length (FL) RB proteins: WT-FL (lane 1),
PSM.2S-FL (lane 2), PSM.2T-FL (lane 3), and PSM.4-FL (lane 4). The transfected cells were metabolically labeled
with [
P]phosphoric acid, lysed, and RB was
immunoprecipitated with anti-RB antibodies. Immunocomplexes were
recovered, resolved by 7.2% (lanes 1-4) SDS-PAGE, and
transferred to Immobilon-P. RB was detected by autoradiography (
P, upper panel), followed by immunoblotting with
anti-RB (
RB, lower panel)
antibodies.
Hyperphosphorylated PSM.2T Binds T-Ag
Because WT
and PSM.2T were both hyperphosphorylated in vivo (Fig. 6A, lanes 1 and 2), we compared
their binding to c-Abl, E2F, and T-Ag (Fig. 6, B-D).
Since PSM.2S was not hyperphosphorylated in vivo, it was not
used in the subsequent assays. The hyperphosphorylated WT and PSM.2T
could not bind to c-Abl (Fig. 6B), nor did they bind to
E2F-1 (Fig. 6C). In contrast, the hyperphosphorylated
bands of PSM.2T could bind to T-Ag (Fig. 6D). These
results were consistent with those obtained with in vitro phosphorylated RB, showing that phosphorylation at Thr-821/826 is
only required for the inhibition of T-Ag binding. Since T-Ag interacts
with RB through an LXCXE motif, we tested whether
phosphorylation of Thr-821/826 was required to regulate the binding of
other LXCXE containing proteins, e.g. E7 and
Elf-1. As with T-Ag, the phosphorylated WT RB did not bind to E7 or
Elf-1 (Fig. 6, E and F, lane 1), while the
phosphorylated upper bands of PSM.2T did bind to these two proteins (Fig. 6, E and F, lane 2). Thus,
phosphorylation cannot inhibit the LXCXE binding
function of RB when Thr-821/826 are mutated.
Figure 6:
Hyperphosphorylated PSM.2T binds to
LXCXE containing proteins. A, in vivo phosphorylatated WT and PSM.2T RB. C33-A cells were cotransfected
with cyclin A and WT-FL (lane 1) and PSM.2T-FL (lane
2). Lysates from the transfected cells were immunoprecipitated
with anti-RB antibody, and resolved by 7.2% SDS-PAGE. RB proteins were
then detected by anti-RB immunoblot. B, hyperphosphorylated
PSM.2T does not bind c-Abl. Lysates from C33-A cells transfected with
WT-FL (lane 1) and PSM.2T-FL (lane 2) were applied to
immobilized GST-Abl (containing the SH2 and tyrosine kinase domains).
Proteins bound to GST-Abl were recovered and resolved on 7.2% SDS-PAGE.
The RB proteins were then visualized by anti-RB immunoblot. C,
hyperphosphorylated PSM.2T RB does not bind E2F-1. The full-length RB
proteins (WT-FL, lane 1, and PSM.2T-FL, lane 2)
expressed in C33-A cells were used in binding reactions with GST-E2F-1,
immobilized on GSH-agarose. Proteins bound to E2F-1 were recovered and
resolved by 7.2% SDS-PAGE. RB was visualized by anti-RB immunoblot. D, in vivo hyperphosphorylated PSM.2T binds T-Ag.
Both the WT and PSM.2T (lanes 1 and 2) proteins were
expressed in C33-A cells, and used in binding reactions with T-Ag. The
fraction bound to T-Ag was recovered and resolved by 7.2% SDS-PAGE. RB
bands were then revealed by immunoblotting with anti-RB antibodies. E, in vivo hyperphosphorylated PSM.2T binds
HPV-16-E7. The WT and PSM.2T (lanes 1 and 2) proteins
coexpressed with cyclin A were used in binding reactions with GST-E7.
The fraction bound to E7 was recovered and resolved by 7.2% SDS-PAGE.
RB bands were revealed by immunoblotting with anti-RB antibodies. F, in vivo hyperphosphorylated PSM.2T binds Elf-1. The WT and
PSM.2T (lanes 1 and 2) proteins produced in C33-A
cells were assayed for binding to GST-Elf-1. RB proteins bound to Elf-1
were recovered and resolved by 7.2% SDS-PAGE. RB was detected by
anti-RB immunoblot.
To determine whether
the GST pull-down assays reflected the regulation of T-Ag binding
inside the cell, we performed coimmunoprecipitation. In C33-A cells
cotransfected with T-Ag and RB, only the underphosphorylated form of WT
was coprecipitated with T-Ag (Fig. 7A, lane 1). In
contrast, the hyperphosphorylated PSM.2T bands were brought down by
anti-T-Ag immunoprecipitation (Fig. 7A, lane 2).
Similar results were also obtained when WT and PSM.2T proteins were
expressed in COS cells and phosphorylated by the endogenous Cdk/cyclins (Fig. 7B, lanes 1 and 2). Thus, PSM.2T binds
to T-Ag irrespective of its phosphorylation status.
Figure 7:
Hyperphosphorylated PSM.2T
coimmunoprecipitates with T-Ag. A, C33-A cells were
cotransfected with T-Ag, cyclin A, and the indicated RB expression
plasmid: WT-FL (lane 1) and PSM.2T-FL (lane 2).
Lysates were either immunoprecipitated with anti-T-Ag (
T-Ag, lower panel) or anti-RB (
RB, upper panel)
antibodies. The recovered proteins were resolved by 7.2% SDS-PAGE. The
RB bands were visualized by immunoblotting with anti-RB antibodies. B, COS cells were transfected with WT-FL (lane 1) or
PSM.2T-FL (lane 2) and used for the coimmunoprecipitation of
RB with T-Ag. Lysates from the cells were either immunoprecipitated
with anti-T-Ag (
T-Ag, lower panel) or anti-RB (
RB, upper panel) antibodies. The recovered proteins were resolved
by 7.2% SDS-PAGE and RB was visualized by
immunoblotting.
Growth Suppression by PSM RB
Phosphorylation of RB
has been correlated with the inactivation of its growth suppression
function in SAOS-2 cells(8) . Exogenous RB does not become
phosphorylated in SAOS-2 cells and causes a cell cycle block at
G
/S which generates growth-arrested flat cells.
Cotransfection of cyclin A can drive RB phosphorylation in SAOS-2 cells
and alleviate the cell cycle block(8) . In keeping with
previous reports, WT RB gave rise to numerous flat cells, and the
number of flat cells was reduced 8-fold with the cotransfection of
cyclin A (Table 2). PSM.2T induced a similar number of flat cells
and the number was reduced 6-fold by the cotransfection of cyclin A.
With PSM.2S, however, cyclin A only caused a 1.6-fold reduction in the
number of flat cells (Table 2). To determine if PSM.2S could be
inactivated by other means, we cotransfected it with T-Ag (Table 2). The wild type RB and PSM.2S were both sensitive to
T-Ag, which caused a 33-fold reduction in the number of flat cells.
Cotransfection with cyclin A drove the phosphorylation of WT and PSM
RB, and the relative level of phosphorylation was similar to that
observed with C33-A cells ( Fig. 5and Table 1B). PSM.2S
was poorly phosphorylated in SAOS-2 cells, and this might account for
its resistance to the inactivation by cyclin A.
DISCUSSION
Under conditions where every Cdk consensus site is
phosphorylated, we have shown that in vitro phosphorylation
does inhibit the protein binding function of RB. Using mutants lacking
specific Cdk phosphorylation sites, we also demonstrated that different
Cdk sites are required for inhibiting distinct RB protein binding
activities. Specifically, Ser-807/811 is required for the inhibition of
c-Abl binding, while phosphorylation of Thr-821/826 is required for the
inhibition of binding to T-Ag, Elf-1, and E7 (all of which contain the
LXCXE motif). However, none of these sites are
required for the inhibition of E2F binding. We find that the mutation
of Ser-807/811 prevents the efficient phosphorylation of RB in
vivo, and cyclin A cannot overcome the growth suppressing activity
of this mutant in SAOS-2 cells. In contrast, mutation of Thr-821/826
does not prevent RB hyperphosphorylation nor the inactivation of its
growth suppressing activity by cyclin A.
Regulation of the A/B Pocket by Phosphorylation
Both
T-Ag and E2F interact with RB through the A/B pocket. However, the
LXCXE motif that mediates the T-Ag binding is not
found in E2F(12, 13) . Furthermore, the CRII fragment
of E1A, which contains the LXCXE motif, cannot
disrupt the RB/E2F complex(28) . Thus, the A/B pocket of RB may
contain two binding sites, one for the LXCXE motif
and another for E2F. This idea is consistent with our finding that the
regulation of T-Ag and E2F binding to RB requires different Cdk
phosphorylation sites. Phosphorylation of Thr-821/826 is required for
the disruption of the T-Ag
RB complex. In addition to T-Ag, we
have found that the inhibition of binding to Elf-1 and E7, both of
which contain the LXCXE motif, also required
Thr-821/826. This finding strongly supports that the
LXCXE binding site is regulated by Thr-821/826
phosphorylation. We cannot determine if both threonine residues or only
one of them is required for the regulation of LXCXE
binding. Single site mutants would have to be constructed to resolve
this issue. Since mutation of Thr-821/826 does not interfere with the
regulation of E2F binding, other phosphorylation sites must be
sufficient to disrupt the A/B pocket function that mediates E2F
interaction. It is interesting to find that the phosphorylation sites
required for the inhibition of LXCXE binding are
outside of the minimal A/B pocket domain. We could envision at least
two possible mechanisms for these phosphothreonines to inhibit the A/B
pocket activity. The phosphorylated threonine may directly bind to the
A/B pocket and sterically hinder the binding of LXCXE
containing proteins. Alternatively, phosphorylation at Thr-821/826 may
result in a conformational change that can lead to the disruption of
the LXCXE binding structure in the A/B pocket.
Inactivation of RB Growth Suppression by
Phosphorylation
The growth suppression function of RB depends on
its ability to assemble protein complexes(16) . This concept is
supported by three lines of evidence: (a) both the large A/B
and the C pockets of RB are required for growth suppression, (b) the A/B and C domains of RB do not function in-trans, and (c) the A/B or C domain fragments can act as dominant
interfering mutants to disrupt RB function. According to this model,
any phosphorylation event that inhibits one of the three protein
binding functions should be sufficient to inactivate the growth
suppression function of RB. This could explain the observation that
PSM.2T-RB, despite the lack of two major phosphorylation sites, can
still be inactivated by cyclin A. The phosphorylated PSM.2T, as shown
here, retains the ability to bind the LXCXE motif,
but cannot suppress SAOS-2 growth. Presumably this is because the c-Abl
and the E2F binding activities are still inactivated by phosphorylation
in this mutant, and therefore the phosphorylated PSM.2T-RB cannot
assemble protein complexes. Alternatively, the LXCXE
binding function may not be relevant for the suppression of SAOS-2
cells. The present data does not rule out the possibility that
PSM.2T-RB may act as a dominant growth suppressor in other cell types.
Role of Partially Phosphorylated RB
If partial
phosphorylation of RB can disrupt its growth suppression function, why
then design multiple phosphorylation sites in RB? One possibility is
that the Cdk-mediated phosphorylation is directed at regulating what
types of complexes RB can assemble. The protein complexes formed on
partially phosphorylated RB may be important in fine-tuning regulatory
events during cell cycle progression. There is evidence that RB
phosphorylation occurs in a stepwise manner during G
progression. Two-dimensional phosphotryptic mapping of RB in
synchronized cells has revealed sequential loading of specific
phosphorylation sites as cells progress from quiescence to S-phase (29) . A conceivable consequence of stepwise phosphorylation
may be as follows: if phosphorylation occurs at sites that inhibit E2F
binding but not at Thr-821/826, this event would release E2F and make
more RB available for binding to the cellular LXCXE
containing proteins. This might be a way for Cdk to orient the A/B
pocket of RB toward specific cellular targets. Partial phosphorylation
of RB might also lead to other modulations of RB binding proteins. For
example, phosphorylation of Ser-807/811, but not at other sites, would
activate the c-Abl tyrosine kinase which could then phosphorylate
proteins still bound to the A/B pocket. This is a possible mechanism
for Cdk to activate the tyrosine phosphorylation of a specific nuclear
protein. The selective, stepwise phosphorylation of RB may provide a
continuous modulation of RB-assembled complexes to alter their
activities throughout the cell cycle.