Discovery of a Regulatory Motif That Controls the Exposure of
Specific Upstream Cyclin-dependent Kinase Sites That
Determine Both Conformation and Growth Suppressing Activity of pRb*
Barbara
Driscoll
,
Anne
T'Ang,
Yi-Hui
Hu,
Chun Li
Yan,
Yue
Fu,
Yi
Luo,
Kai Jin
Wu,
Shumin
Wen,
Xiang-He
Shi,
Lora
Barsky,
Kenneth
Weinberg,
A. Linn
Murphree, and
Yuen Kai
Fung§
From the Divisions of Hematology/Oncology, Ophthalmology,
Pathology, and Research Immunology/Bone Marrow Transplant, Childrens
Hospital Los Angeles, and the Departments of Pediatrics, Microbiology,
Ophthalmology, Pathology, and Immunology, University of Southern
California, School of Medicine, Los Angeles, California 90027
 |
ABSTRACT |
The conformation and activity of pRb,
the product of the retinoblastoma susceptibility gene, is dependent on
the phosphorylation status of one or more of its 16 potential
cyclin-dependent kinase (cdk) sites. However, it is not
clear whether the phosphorylation status of one or more of these sites
contributes to the determination of the various conformations and
activity of pRb. Moreover, whether and how the conformation of pRb may
regulate the phosphorylation of the cdk sites is also unclear. In the
process of analyzing the function and regulation of pRb, we uncovered
the existence of an unusual structural motif, m89 (amino acids
880-900), the mutation of which confers upon pRb a hypophosphorylated
conformation. Mutation of this structural domain activates, rather than
inactivates, the growth suppressor function of pRb. In order to
understand the effect of the mutation of m89 on the phosphorylation of
cdk sites, we identified all the cdk sites (Thr-356, Ser-807/Ser-811, and Thr821) the phosphorylation of which drastically modify the conformation of pRb. Mutation of each of these four sites alone or in
combinations results in the different conformations of pRb, the
migration pattern of which, on SDS-polyacrylamide gel electrophoresis, resembles various in vivo hypophosphorylated forms. Each of
these hypophosphorylated forms of pRb has enhanced growth suppressing activity relative to the wild type. Our data revealed that the m89
structural motif controls the exposure of the cdk sites Ser-807/Ser-811 in vitro and in vivo. Moreover, the m89 mutant
has enhanced growth suppressing activity, similar to a mutant with
alanine substitutions at Ser-807/Ser-811. Our recent finding, that the
m89 region is part of a structural domain, p5, conserved antigenically
and functionally between pRb and p53, suggests that the evolutionarily
conserved p5 domain may play a role in the coordinated regulation of
the activity of these two tumor suppressors, under certain growth conditions.
 |
INTRODUCTION |
The product of the retinoblastoma susceptibility gene, RB-1,
exists in multiple conformations. The various forms of pRb, even after
denaturation, maintain their conformational differences that are
readily discerned by
SDS-PAGE.1 The stability of
particular localized conformation of certain polypeptides under the
denaturing conditions of SDS-PAGE has previously been described (1, 2)
and is perhaps a result of hydrophobic intramolecular interaction. At
least five different forms of pRb isolated from an asynchronous culture
can be discerned by SDS-PAGE. While there is no doubt that these
different conformations of pRb are a SDS-PAGE gel artifact, previous
studies have clearly showed that they correspond to the phosphorylation
status of the pRb.
Multiple forms of heavily phosphorylated pRb appear in late
G1 and S phases of the cell cycle. In quiescent and
differentiating cells, pRb is hypophosphorylated and migrates fastest
in SDS-PAGE (3-5) compared with other forms observed in late
G1 and S. This form of pRb is also observed in cells at the
M and early G1 phases, when pRb is dephosphorylated (6).
There are 16 Ser/Thr-Pro motifs on pRb that are potential
cyclin-dependent kinase (cdk) targets. Functional evidence
that the phosphorylation of some or all of these cdk sites regulates
the conformation and activity of pRb comes from the observation that
ectopic expression of cyclins A, D, and E can overcome the growth
suppression effect of pRb (7, 8). Other in vitro and
in vivo experiments have shown that various viral and
cellular proteins bind preferentially to the hypophosphorylated forms
of pRb (Refs. 10 and 11; reviewed in Ref. 9). For example, the binding
of pRb to the SV40 large T antigen is specifically affected by the
phosphorylation status of Thr-821/Thr-826, while the phosphorylation
status of Ser-807/Ser-811 controls the assembly of the pRb:c-Abl
complex (11). Phosphorylation by cdks, in turn, leads to the disruption
of protein complexes formed between pRb and its targets. Together,
these data suggest that the conformation and activity of pRb are
regulated by the phosphorylation status of its cdk sites.
While it is clear that the cell cycle-dependent
phosphorylation of the 16 potential cdk sites regulates the
conformation of pRb, there is surprisingly little information on the
relationship between the phosphorylation status of a particular site
and a particular conformation. To date, the only cdk sites that have been reported to have an effect on a particular conformation of pRb are
those of the combined sites Ser-807/Ser-811 (11-14), or when most cdk
sites were mutated (12), although pRb clearly can exist in multiple
conformations. It is not known how many of the cdk sites are
phosphorylated in any given form of pRb and which of these sites
contribute to the determination of a particular conformation. It is
also unclear whether these cdk sites act independently or
synergistically in determining the conformation of pRb, and whether the
resulting conformational changes alter the growth suppressing activity
of pRb.
There are several lines of evidence which suggest that the
phosphorylation of pRb occurs in a stepwise manner as cells progress through the cell cycle. As synchronized cell populations traverse the
cell cycle, progressive changes in the phosphorylation of pRb can be
discerned by one-dimensional SDS-PAGE analysis (3, 4, 15) as well as by
two-dimensional phosphopeptide analyses, in which distinct pRb
phosphorylation patterns emerge at the G1 and S phases (4,
14, 16, 17). How this stepwise phosphorylation of particular cdk sites
is regulated is unknown at present, but it could be due to a change in
the expression of cdk enzyme activities or the conformation of the pRb
substrate which may control the exposure of the cdk sites, or both. The
fluctuation in the level of cdk-regulating cyclins at different phases
of the cell cycle is clearly one mechanism for the stepwise regulation
of phosphorylation of particular cdk sites. On the other hand, previous
data have shown that the phosphorylation of pRb is also dependent on
the conformation of pRb itself. Many naturally occurring mutations within the N terminus, C terminus, and the A/B pocket domains of pRb
have been reported to render pRb refractory to phosphorylation (18-21). Naturally occurring point mutations at Ser-567 and Cys-706 have a drastic effect on the conformation of pRb, as shown by the loss
of ability to bind various target proteins, or to be phosphorylated.
Taken together, these data suggest that the phosphorylation of pRb is
also regulated by its conformation.
In our attempts to analyze the function and regulation of pRb, we
performed a detailed analysis of the domains that are associated with a
particular function and/or regulation by creating a panel of pRb
mutants in which certain amino acid residues in a 20-amino acid stretch
were mutated. The phosphorylation patterns of the resulting mutants and
their ability to suppress cell growth were analyzed. Consistent with
previous reports, most mutations in the A/B pocket region resulted in
the inactivation of pRb, as measured by the inability of the
ectopically expressed mutant to suppress cell growth. In addition to
these inactivating mutations, most mutations in other regions have no
apparent effect on the growth suppression function of pRb. However, in
the course of these analyses, we uncovered the existence of an unusual
structural motif, m89 (amino acid residues 880-900), the mutation of
which confers upon pRb a hypophosphorylated conformation and an
enhanced growth suppressor activity.
To identify the cdk sites affected by m89, each of 15 cdk sites
(Ser-567 excluded) was individually mutated and the effect on the
phosphorylation status and conformation of pRb analyzed. Our results
revealed that there are four cdk sites, Thr-356, Ser-807/Ser-811, and
Thr-821, the phosphorylation status of which exerts a drastic change in
the conformation of pRb independent of other sites. Moreover, all five
different forms of pRb isolated from an asynchronous culture,
resolvable by SDS-PAGE, can apparently be accounted for by the
phosphorylation status of one or more of these four sites.
Data presented below shows that the integrity of the m89 structural
motif determines the exposure of two of these four cdk sites, namely
Ser-807 and Ser-811 in vitro and in vivo. Bladder carcinoma 5637 cells expressing both a transfected cyclin A and m89
mutant pRb were blocked in cell cycle progression to a greater extent
than with the wild type pRb. The m89 motif is therefore a structural
domain, other than cdk sites, the mutation of which activates, rather
than inactivates, the function of pRb.
 |
EXPERIMENTAL PROCEDURES |
Construction of Expression Plasmids for pRb cDNA and Its
Mutants--
Phosphorylation site mutants in which serines and
threonines were changed to alanine, were subcloned into pBluescriptSK+, to generate pBSpRbmS/TXXXA, where XXX is the
amino acid residue number of the cdk sites. Double mutants were created
at sites Ser-608/Ser-611 and Ser-807/Ser-811. A triple mutant was
created at Thr-356/Ser-807/Ser-811, while a quadruple mutant was
created by combining the triple mutant with the Thr-821 mutant to
create Thr-356/Ser-807/Ser-811/Thr-821. The regulatory site mutant
pBSpRbm89 was created by changing the amino acids 880-900 of the m89
region EGSDEADGSKHLPGESKFQQK to
EASAEVDASIHLPGESKFQQK.
The plasmids peGFP/CycA/pRb, peGFP/CycA/pRbm89,
peGFP/CycA/pRbmS807S811, peGFP/CycA/pRbmT356/S807/S811, and
peGFP/CycA/pRbmT356/S807/S811/T821 were constructed by subcloning the
pRb cDNAs under the control of the immediate early promoter of
cytomegalovirus (pCMV) into peGFP/CycA containing a pCMV-cyclin A. The
coding sequence for the enhanced green fluorescence protein (eGFP) was
linked to pRb cDNA via an internal ribosomal entry site element
(IRES, CLONTECH).
Cell Cycle Analysis--
Human bladder carcinoma 5637 cells were
seeded into T25 flasks at 20% confluence. The following day, cells
were transfected with 5 µg of the selected plasmid, together with
LipofectAMINE, according to manufacturer's instructions (Life
Technologies, Inc.). Control cells were mock-transfected with
LipofectAMINE alone. After 6 h, leftover DNA and LipofectAMINE
were removed by washing the cells with phosphate-buffered saline. Cells
cultured in RPMI 1640 medium in the presence or absence of serum for
56 h at 37 °C were harvested by trypsinization, fixed in 4%
paraformaldehyde in phosphate-buffered saline, and stained with
propidium iodide. The cells were then FACS sorted into GFP-positive and
-negative populations using a Becton Dickinson FACScan equipped with a
488-nm argon laser as light source and CellQuest software. For these experiments, 100,000 events were acquired and the non-clumped cells
were gated for analysis on a second dual parameter display with DNA
(linear red fluorescence) on the x axis and FITC-eGFP (log
green fluorescence) on the y axis. Negative and positive parameters were set with cells transfected with eGFP plasmid or exposed
to LipofectAMINE alone. For cells transfected with other plasmids,
GFP-positive events were analyzed for DNA content and cell cycle
positions using ModFit LT 2.0 DNA analysis software (Verity House
Software, Topsham, ME).
Phosphorylation Studies--
Immunoprecipitations were performed
as described previously (3). 35S-Labeled wild type and
mutant pRb proteins were produced by in vitro transcription
and coupled translation of the pBluescript/pRb plasmids using the TNT
T7 coupled reticulocyte lysate system (Promega), together with
Expre35S35S (labeled at the methionine and
cysteine residues). For immunoprecipitation, reticulocyte lysate was
diluted in EBC buffer (125 mM NaCl, 40 mM Tris
base, pH 8.0, 0.5% Nonidet P-40) containing proteinase and phosphatase
inhibitors (100 mM phenylmethylsulfonyl fluoride, 10 mM each of aprotinin, leupeptin, pepstatin, and
-glycerol phosphate, 20 mM NaF, and 0.1 mM
NaVO4), antibody RB1-Ab 2-3 (3), and protein
A-agarose (Sigma). The immunoprecipitated proteins were resolved
by 6% SDS-PAGE.
For in vitro kinase reactions, a crude extract of pRb kinase
was prepared and used as described previously (3).
107 Sf9 cells co-infected with cyclin A
and cdk2 baculoviruses were lysed in kinase reaction buffer (KRB: 20 mM Tris base, pH 7.5, 20 mM MgCl2,
10 mM
-mercaptoethanol plus proteinase and phosphatase inhibitors) supplemented with 150 mM NaCl and 0.5% Nonidet
P-40. Clarified extracts were used to phosphorylate wild type and
mutant pRb proteins immunoprecipitated from the reticulocyte mixture. All reactions contained ATP at a final concentration of 100 mM.
For two-dimensional phosphopeptide analysis, pHu
AcprpRbPQ,
pHu
AcprpRbm89, and pHu
AcprpRbmS807/S811 were separately
transfected into Saos-2-AT (14, 22). Neomycin-resistant clones were
selected for in G418 and grown in RPMI 1640 medium supplemented with
10% fetal bovine serum and antibiotics at 37 °C. pRb phosphorylated in vivo was immunoprecipitated from Saos-2 transfectants
metabolically labeled with
[32P]H3PO4. Immunoprecipitates
were resolved by 8% SDS-PAGE. Chymotryptic digestion of pRb and
analysis of phosphopeptides were performed as described previously
(14). Briefly, proteins were electrophoretically transferred to a
nitrocellulose filter, which was exposed to x-ray film. Bands
corresponding to pRb were excised, and the protein, extracted and
immunoprecipitated from 5 × 106 cells, was digested
with 20 µg of chymotrypsin (Sigma) in 200 µl of 50 mM
ammonium bicarbonate, pH 7.3, at 37 °C for 18 h. Chymotryptic peptides were dried, washed, and oxidized using performic acid. Following oxidation, peptides were washed extensively, first with water, then with thin layer electrophoresis buffer (TLE buffer: 2.2%
formic acid, 7.8% acetic acid, pH 1.9). For an accurate comparison between samples, the same number of cpm of each sample was applied. Peptides were spotted onto a thin layer cellulose plate (Kodak) and
electrophoresed in the first dimension in TLE buffer, for 1.5 h,
using a Hunter thin layer electrophoresis apparatus (HTLE-7000, C.B.S.
Scientific, San Diego, CA). Plates were dried, then chromatographed in
the second dimension using phosphochromatography buffer
(n-butanol:pyridine:acetic acid:water, 5:3:1:4 by volume).
Dried plates were exposed to x-ray film.
 |
RESULTS |
Discovery of the m89 Motif Which Confers upon pRb a
Hypophosphorylated Conformation--
To understand the function and
regulation of pRb, we performed in vitro mutagenesis
throughout the RB-1 cDNA. Forty-seven different amino acid
substitution mutants, each covering 20 amino acid residues, were
created, and the effect of the mutation on the function and regulation
of phosphorylation of pRb analyzed using different cell lines. As
expected, mutations in the pocket region resulted in the inactivation
of pRb function.
In order to understand the effect of the mutations on the regulation of
pRb phosphorylation, we made use of a Saos2 variant cell line
(Saos-2AT), which is resistant to growth inhibition by ectopically
expressed pRb (22-24). Cell lines harboring the transfected mutants
were isolated, and the in vivo phosphorylation status of the
ectopically expressed pRb in each was analyzed. In the process, we
encountered a mutant, pRbm89, which exhibited a different mobility by
SDS-PAGE than wild type pRb. As is shown in Fig.
1A, wild type pRb isolated
from an asynchronous cell population of Saos2pRbPQ (lane
1) multiple forms in a one-dimensional SDS-PAGE. In
contrast, the mutant pRb isolated from an asynchronous cell population
of Saos2pRbm89 exhibited only hypophosphorylated forms (lane 2). Since pRbm89 protein was isolated from
an asynchronous population, this observation suggests that the mutant
form of pRb assume a hypophosphorylated conformation, even at the S
phase of the cell cycle when wild type pRb is normally fully
phosphorylated. Indeed, pRbm89 protein isolated from a cell population
growth arrested at G1/S with alphidicolin remains in the
same hypophosphorylated conformation (data not shown).

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Fig. 1.
Analysis of the effect of mutation of m89 on
the phosphorylation status of pRb. Panel A, SDS-PAGE
profiles of immunoprecipitated wild type pRb (lane
1) and m89 mutant pRbm89 (lane 2),
extracted from [35S]methionine metabolically labeled
randomly growing cultures of transfected Saos2 (AT) cells. Panel
B, SDS-PAGE profiles of immunoprecipitated wild type pRb
(lanes 1 and 3) and m89 mutant pRbm89
(lane 2) proteins generated by in
vitro translation with [35S]methionine. The pRb were
either not phosphorylated (lane 1) or
phosphorylated in vitro (lanes 2 and
3) using a kinase preparation from Sf9
cells co-infected with baculoviruses expressing cyclin A and
cdk2.
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To understand if the hypophosphorylated conformation of the pRbm89
mutant is due to mutation at the m89 region or due to a peculiar lack
of a specific cdk activity in the Saos2pRbm89 cell line, we analyzed
the protein in vitro phosphorylation pattern of the pRbm89.
In vitro translated mutant and wild type pRb were kinased
with either cyclin A/cdk2, cyclin D2/cdk4, or cyclin E/cdk2. As is
shown in Fig. 1B, under optimized kinasing conditions where unphosphorylated wild type pRb (lane 1) became
fully phosphorylated (lane 3), the kinased pRbm89
still maintains the hypophosphorylated conformation (Fig.
1B, lane 2) with an apparent molecular
mass midway between the unphosphorylated and the hyperphosphorylated forms. The fact that the hypophosphorylated conformation of pRbm89 is
observed both in vivo in the S phase, and in
vitro under optimized conditions when wild type pRb is fully
phosphorylated, suggests that it is an intrinsic property of the m89
mutant and not the result of suboptimal cdk activity.
There are several possible explanations for the observed conformation
of pRbm89. One is that all sites on the protein are fully
phosphorylated, but that mutation at m89 has somehow altered conformation of the phosphorylated form of pRb. Alternatively, it is
possible that the mutation at m89 has caused a conformational change in
pRb, such that particular phosphorylation sites, critical for the
determination of the conformation of pRb, are not exposed to the
kinase. To distinguish between these two possibilities, it is necessary
to have an understanding of the relationship between the conformation
of pRb and the phosphorylation status of the cdk sites. By identifying
the cdk sites that control the migration pattern of the various forms
of pRb (Fig. 2A,
lane 1), it should be possible to determine if
the mutation at m89 has altered the exposure of the cdk sites that
confer the particular conformation of pRbm89. A detailed analysis of
the cdk sites and their contribution to the conformations of pRb was
therefore performed.

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Fig. 2.
Detailed analysis of the relationship between
the phosphorylation status of individual cdk sites and the conformation
of pRb. Panel A, SDS-PAGE profiles of
immunoprecipitated wild type pRb extracted from a random culture of
Saos-2 (AT) cells expressing a transfected wild type pRb plasmid
(lane 1). The five most easily identifiable forms
were designated forms 1-5, with form 1 being the fastest migrating,
least phosphorylated form and form 5 being the most heavily
phosphorylated, slowest migrating form. Wild type pRb extracted from
cells growth-arrested by serum starvation is included for comparison
(lane 2). Panel B, SDS-PAGE profiles
of immunoprecipitated wild type and cdk site mutant pRb, labeled with
[35S]methionine during in vitro translation,
and kinased using a combination cyclin A/cdk2 kinase extracted from
recombinant baculovirus-infected Sf9 cells. Wild type
unphosphorylated pRb (lanes 1 and 14)
and fully phosphorylated pRb (lanes 2 and
13) pRb cdk mutants at Ser-249 (lane
3), Thr-252 (lane 4), Thr-356
(lane 5), Thr-373 (lane 6),
Ser-608/Ser-612 (lane 7), Ser-788
(lane 8), Ser-795 (lane 9),
Ser-807/Ser-811 (lane 10), Thr-821
(lane 11), and Thr-826 (lane
12) were kinased under identical conditions and the
immunoprecipitated proteins resolved by SDS-PAGE. Panel C,
schematic representation of pRb, showing the location of 16 putative
cdk target sites with the four cdk sites, which affect pRb conformation
shown in black. Pocket regions A and B, including the
inactivating point mutation site Ser-567, are shaded in
gray, and the site of the m89 mutation is marked in
black.
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Detailed Analysis of the Relationship between the Phosphorylation
Status of Individual cdk Sites and the Conformation of pRb--
To
understand the status of phosphorylation of each of the cdk sites in
the various forms of pRb, we mutagenized the serine or threonine
residues of individual cdk sites into alanine. In choosing the cdk
sites for detailed analysis, Thr-5, Ser-230, and Ser-780, which have
been shown not to be phosphorylated in vivo (25), and
Ser-567, the mutation of which reportedly inactivates pRb (19), were
excluded from analysis. The remaining 12 sites: Ser-249, Thr-252,
Thr-356, Thr-373, Ser-608, Ser-612, Ser-788, Ser-795, Ser-807, Ser-811,
Thr-821, and Thr-826, which have been demonstrated to be phosphorylated
in vivo (25-27), were analyzed. To show that mutations at
these 12 cdk sites do not inactivate the function of pRb, corresponding
mutants were subcloned into both the pHu
Acpr-1-neo and the pAcYM1
vectors for growth suppression and protein binding analysis. Ectopic
expression of these mutants driven by the human
actin promoter
resulted in suppression of the growth of Saos-2 cells (ATCC HTB
85) (data not shown). In addition, all of these mutants, expressed as
recombinant baculoviruses in Sf9 cells, retain their
ability to bind the SV40 large T antigen and the cellular proteins
c-Abl and E2F-1 in vitro (data not shown). These results
indicated that, unlike the mutation at Ser-567, none of these
mutations were inactivating.
To understand the effect of the mutation of the cdk sites on the
conformation of pRb, we compared the migration pattern of in
vitro translated pRb mutants to the various forms of pRb extracted from cell culture, on SDS-PAGE. As is shown in Fig. 2A
(lane 1), wild type pRb protein extracted from an
asynchronous cell culture can be fractionated into five distinct forms.
We designated these as forms 1-5, with form 1 being the most
underphosphorylated, fastest migrating form and form 5 being the fully
phosphorylated and slowest migrating form. As expected, form 1 pRb is
the only form extracted from cells growth-arrested by serum starvation (Fig. 2A, lane 2).
For in vitro analysis, wild type and cdk site mutants of
pRb, generated by in vitro translation, were kinased under
optimized conditions and their migration analyzed by one-dimensional
SDS-PAGE. As expected, unphosphorylated wild type pRb migrates as form
1 (Fig. 2B, lanes 1 and 14)
and is converted into the slowest migrating form 5 (lanes
2 and 13) by the kinasing reaction. Differences in the migration pattern of the optimally phosphorylated cdk site mutants were reproducibly observed (Fig. 2B,
lanes 3-12). Of these, mutations at Thr-356
(lane 5), Ser-807/Ser-811 (lane
10), and Thr-821 (lane 11) resulted in
the most noticeable difference in the migration pattern. It should be
noted that these pRb mutants were otherwise fully phosphorylated at all
the other cdk sites, as determined by two-dimensional peptide analysis
using the wild type pattern as a standard (Fig. 4B). Thus,
if either Thr-356 (lane 5) or Thr-821
(lane 11) is not phosphorylated, the otherwise fully phosphorylated pRb mutant migrates to a slightly faster position,
corresponding to form 4 in Fig. 2A. Mutation of
Ser-807/Ser-811 causes a more drastic effect on pRb conformation (Fig.
2B, lane 10), resulting in a migration
pattern similar to that of form 3. Thus, mutation at Thr-356, which is
N-terminal to the pocket region, or Ser-807/Ser-811 or Thr-821, which
are C-terminal to the pocket region, can individually cause
conformational changes independent of all other sites. While Thr-356
was previously reported to play no role in the determination of pRb
conformation (12), the data provided here clearly show that Thr-356 has
a drastic effect on the conformation of the N-terminal domains upstream of the pocket region. The slight but reproducible difference in the
migration of these mutants is consistent with changes being individual
and local.
Mutation of serine/threonine to alanine at Ser-249 (lane
3), Thr-252 (lane 4), Thr-373
(lane 6), Ser-608/Ser-612 (lane
7), Ser-788 (lane 8), Ser-795
(lane 9), and Thr-826 (lane
12) has no obvious effect on the conformation of pRb when
all other cdk sites are phosphorylated. The slight variations in the
migration patterns (presumably due to gel drying artifacts) of some of
these sites were not reproducible from experiment to experiment. The only consistent changes were those of the four sites described above
(summarized in Fig. 2C).
As these changes appear to be independent of each other, we
investigated the possibility that the conformational changes are additive by performing an analysis on pRb mutated at more than one
site. The resulting data showed that the conformational changes brought
about by mutation at Thr-356 and Ser-807/Ser-811 appear to be additive,
such that a mutant encompassing all three sites (Fig.
3A, lane
3) migrates faster than either of the mutants alone (lane 2), to a position similar to that of form
2. Thus, the conformation of pRb lacking phosphorylation at Thr-356,
located in front of the pocket region, appears to be independent and
additive to the conformation caused by mutation at Ser-807 and Ser-811,
located behind the pocket region. The conformational change brought
about by mutation at Thr-821 is also additive, in that mutation at all four critical sites (Thr-356, Ser-807/Ser-811, and Thr-821) produces a
form of pRb with a migration pattern similar to that of the unphosphorylated form (form 1) (Fig. 3A, lane
4; see also Fig. 4A, lanes
1 and 2). It should be noted that although there
is an additive change in the conformation in pRb mutated at both Thr-356 and Thr-821 (Fig. 3B, lanes 1 and 2) or Ser-807/Ser-811 and Thr-821 (Fig. 3B,
lanes 5, 6, and 7), the
change is difficult to be resolved satisfactorily by one-dimensional
SDS-PAGE. Thus, what we called form 3 pRb, isolated from an
asynchronous cell population could, in fact, be a mixed pRb population
consisting of pRb unphosphorylated at Ser-807/Ser-811 with or without
phosphorylation at Thr-821. Likewise, form 4 could be composed of a
mixture of pRb unphosphorylated Thr-356, Thr-821, or a combination of
the two. In summary, the five forms of pRb observed in vivo
can be accounted for by the lack of phosphorylation at one or more of these four cdk sites, even when all other sites are phosphorylated (summarized in Fig. 3C).

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Fig. 3.
Effect of mutation of multiple
phosphorylation sites on pRb conformation. Panels A and
B, SDS-PAGE profiles of immunoprecipitated wild type and cdk
site mutant pRb, labeled with [35S]methionine during
in vitro translation, and kinased using a combination cyclin
A/cdk2 kinase extracted from recombinant baculovirus-infected
Sf9 cells. Panel A, fully phosphorylated
wild type pRb (lane 1) was compared with fully
phosphorylated mutant pRb mutated at Ser-807/Ser-811 alone
(lane 2), or at both Ser-807/Ser-811 and Thr-356
(lane 3), or at Thr-356, Ser-807/Ser-811, and
Thr-821 (lane 4). Panel B, migration
pattern of kinased Thr-356 mutant (lane 1) was
compared with combination mutants Thr-356/Thr-821 (lane
2), Thr-356/Ser-807/Ser-811 (lane 3),
and to the Thr-356/Ser-807/Ser-811/Thr-821 mutant (lane
4). Migration pattern of the mutant at Thr-821
(lane 5) was compared with pRbmT821/S807/S811
(lane 6) and to pRbmS807/S811 (lane
7). Panel C, a schematic representation of the
four phosphorylation sites on pRb, the modification of which appears to
contribute to alterations in pRb conformation. Numbered
bars represent pRb forms observed when the protein was
extracted from an asynchronous culture (see Fig. 2A). In
form 1, all four sites are unphosphorylated ( ). Form 2 requires that
the combination of Thr-356 and Ser-807/Ser-811 remain unphosphorylated,
while Thr-821 is phosphorylated ( ) (see A). Form 3 is
derived from forms in which either only Ser-807/Ser-811 is
unphosphorylated while all others, including Thr-356, are (see Fig.
2B and panel A of this figure), or when Thr-821 is also unphosphorylated, in combination
with Ser-807/Ser-811 (data not shown). In this case, the
unphosphorylated status of Thr-821 does not have the noticeable
additive effect on conformation that occurs when both Thr-356 and
Ser-807/Ser-811 are both unphosphorylated. Form 4 can be produced by
phosphorylation of all available sites on pRb with the exception of
either Thr-821 or Thr-356 (see Fig. 2B). As mentioned for
form 3, form 4 can also be produced when both Thr-356 and Thr-821 are
unphosphorylated (data not shown), another example of the much less
noticeable additive effect of modification at Thr-821, when only one
other site is also modified. Form 5 corresponds to pRb in which all
available sites, including Thr-356, Ser-807/Ser-811, and Thr-821, are
phosphorylated.
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|

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Fig. 4.
Comparison of the effect of cdk site and m89
mutations by one-dimensional SDS-PAGE migration and two-dimensional
phosphopeptide analysis. Panel A, SDS-PAGE profiles of
immunoprecipitated wild type and cdk site mutant pRb labeled with
[35S]methionine during in vitro translation
and kinased using a combination cyclin A/cdk2 kinase extracted from
recombinant baculovirus-infected Sf9 cells. The fully
phosphorylated version of pRb, in which all four cdk sites (Thr-356,
Ser-807, Ser-811, and Thr-821) are mutated (lane
1), was compared with unphosphorylated wild type
(lane 2), the phosphorylated form of the m89
mutant pRbm89 (lane 3), and to the fully
phosphorylated forms of the mutant Ser-807/Ser-811 (lane
4) and wild type pRb (lane 5).
Panel B, two-dimensional phosphopeptide patterns were
obtained by chymotryptic digestion of wild type and mutant pRb proteins
extracted and immunoprecipitated from transfected Saos2 (AT) (wild
type, pRbm89, and pRbmS788S807S811) cells and TGF -treated MDAMB 231 cells, metabolically labeled with
[32P]H3PO4. For each panel, the
phosphopeptides corresponding to those contributed by cdk sites
Thr-356, Ser-788, Ser-795, Ser-807/Ser-811, and Thr-821, determined by
chymotryptic mapping of cdk site mutants, are circled and
labeled. For the combination cdk site mutant Ser-788/Ser-807/Ser-811
from Saos2pRbm788/807/811C2, mutation at the Ser-788 site had no effect
on either the one- or two-dimensional phosphorylation patterns of
pRbmS807/S811 (data not shown), and therefore, this mutant is used as
an example of the pRbmS807/S811 pattern. For quantitation of
phosphorylation at the four critical sites due to mutation or TGF
treatment, see Table I.
|
|
The m89 Region Regulates the Exposure of the Ser-807/Ser-811 cdk
Sites--
To determine if mutation at the m89 region resulted in a
conformational change that blocks cdk site exposure, we compared the
migration pattern of in vitro phosphorylated pRbm89 to those of the cdk site mutants (Fig. 4A). Our results showed that
the migration pattern of pRbm89 matched that of pRb mutated at
Ser-807/Ser-811 (Fig. 4A, lanes 3 and
4).
To determine if the Ser-807/Ser-811 sites are indeed affected by the
m89 mutation, we performed two-dimensional phosphopeptide mapping
analysis of the in vivo phosphorylation patterns of
individual cdk sites in the wild type pRb and pRbm89 (Fig.
4B). The chymotryptic phosphopeptide containing the
Ser-807/Ser-811 sites was previously mapped using the 807/811 mutant
and the data confirmed with a synthetic peptide corresponding to the
chymotryptic phosphopeptide containing Ser-807/Ser-811 (14). The
chymotryptic phosphopeptides corresponding to Thr-821, Ser-795, and
Thr-356 were mapped using the corresponding mutants in vitro
and in vivo. In order to quantitate the changes in
phosphorylation at these four cdk sites that may have occurred in
pRbm89, x-ray film images of the two-dimensional patterns of wild type
and mutant pRb were analyzed by scanning densitometry. As the
phosphorylation status of Ser-795 showed little variation with growth
conditions, it was used as the standard for comparison between samples.
By normalizing the intensity of the peptide images of Ser-795 from the
wild type and various pRb mutants, the phosphorylation status of the
four cdk sites was compared. The data (Fig. 4B and Table
I) revealed that phosphorylation at
Ser-807/Ser-811 was absent in both the pRbmS788/S807/S811 mutant and in
pRbm89, and was reduced in pRb extracted from TGF
-treated, growth-arrested, MDAMB 231 cells. In contrast, negligible changes were
observed for those peptides containing Thr-356 or Thr-821 in these
mutants, or under TGF
treatment conditions. As such, the data showed
very clearly that the Ser-807/Ser-811 region in the m89 mutant, pRbm89,
is inaccessible to kinases in vivo. In contrast, the
chymotryptic phosphopeptides corresponding to Thr-821, Ser-795, and
Thr-356 (mapped using the corresponding mutants in vitro and
in vivo) were not affected by the m89 mutation. This in vivo result was confirmed when wild type pRb and pRbm89
were phosphorylated in vitro (data not shown).
View this table:
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|
Table I
Phosphorylation site status
Each two-dimensional peptide pattern was analyzed by scanning
densitometry. Values for peptide Ser-795 were normalized to 1.0. Values
for peptides Ser-807/Ser-811, Thr-356, and Thr-821 were assigned values
based on comparison to Ser-795. Values represent mean ± 0.1 for
two separate experiments.
|
|
That the mutation of m89 affects the phosphorylation of Ser-807/Ser-811
specifically is of great interest in view of the fact that the flanking
Thr-821 and Ser-795 sites were not affected. Our previous data revealed
that phosphorylation of Ser-807/Ser-811 were also specifically
down-regulated in cells growth-arrested at the late G1
phase by TGF
treatment (14). An example of this form of endogenous
pRb extracted from TGF
-treated MDAMB 231 cells is included here for
illustration (Fig. 4B). We further showed that the
Ser-807/Ser-811 sites became rapidly phosphorylated once TGF
was
removed and cells progressed into the S phase (14). These data suggest
that pRb unphosphorylated at Ser-807/Ser-811 is in an active form
capable of blocking cell cycle progression. It further suggests that
pRb mutated at the m89 region should be refractory to inactivation by
phosphorylation, since the critical Ser-807/Ser-811 sites are rendered
inaccessible. As such, pRbm89 should be constitutively active. To test
this hypothesis, we compared the growth suppression properties of the
wild type and the mutant pRb.
Comparison of the Ability of Wild Type and Constitutively Active
pRb Mutants to Block Cell Cycle Progression--
To compare the effect
of the mutation of the four cdk sites and the m89 region on the growth
suppressor function of pRb, selected cdk site mutants and the m89
mutant pRbm89 were placed under the control of the CMVp in a plasmid
together with CMVp-cyclin A and the fluorescent marker eGFP. The
resulting plasmids, peGFP, peGFP/CycA, peGFP/CycA/pRb,
peGFP/CycA/pRbm89, peGFP/CycA/pRbmS807/S811,
peGFP/CycA/pRbmT356/S807/S811, and peGFP/CycA/pRbmT356/S807/S811/T821,
were transfected into the human diploid bladder carcinoma cell line,
5637. Cells were cultured for 56 h in the presence or absence of
serum. The harvested cells were then fixed, counterstained with
propidium iodide, and FACS sorted into GFP-positive and -negative
populations. Cell cycle analysis was performed on the GFP-positive
population. In the peGFPtransfected, GFP-positive cells, the effect
of serum deprivation could clearly be seen in the increase in cells in G0/G1, coupled by a marked decrease in the S
phase population. When compared with serum-deprived
GFP-positive cell culture, co-expression of cyclin A with eGFP resulted
in a significant decrease in the G0/G1
population (*, p < 0.0005) while the S and
G2/M populations showed significant increases (**,
p < 0.0005; ***, p < 0.02), even in
the absence of serum (Fig.
5A).

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Fig. 5.
Cell cycle analysis of GFP-positive human
bladder carcinoma 5637 cells expressing wild type or mutant pRb/eGFP
fusion proteins and cyclin A. The percentage of cells at various
cell cycle phases were determined for cells transfected with various
peGFP plasmids. Data represent the mean ± standard deviation for
three separate FACS analyses. Statistical significance was calculated
using Student's t test. Panel A, comparison of
G0/G1, S, and G2/M phase profiles
from populations of peGFP-transfected cells maintained in medium plus
or minus serum, and cells transfected with a plasmid co-expressing
peGFP and cyclin A (peGFP/CycA), also under serum-deprived conditions.
For peGFP/CycA-transfected GFP-positive cells, the decrease in the
G0/G1 population (*, p < 0.0005) and the increase in the S and G2/M populations were
significant (**, p < 0.0005; ***, p < 0.02). Panel B, cell cycle analysis of multiple cdk site and
m89 region pRb mutant-transfected cells. All cell lines were analyzed
following transfection and maintenance in medium without serum with the
exception of sample 1, which controls for the effect of eGFP expression
alone, and represents a random population in the presence of serum.
Results are expressed as the number of cells in S phase following
transfection and treatment, and are considered representative of the
cycling portion of the population, as opposed to cells which may have
become blocked at the G0/G1 or G2/M
phases. No significant decrease in the number of cycling cells was
observed by co-expressing wild type pRb with cyclin A (compare
bars 3 and 4). However, mutation at
the m89 region ( ) or at Ser-807/Ser-811 ( ) did
cause a significant drop in S phase cells when compared with levels
observed in the wild type transfected cells (**, p < 0.01; ***, p < 0.01). Addition of the Thr-356 mutation
to the Ser-807/Ser-811 mutation caused a significant difference in the
cycling population when compared with wild type pRb ( ,
p < 0.005), and this difference was more significant
than the difference between Ser-807/Ser-811 alone and wild type ( ,
p < 0.005; compare with ***, p < 0.01). Addition of a mutation at Thr-821 caused a significant decrease
in the number of cycling cells when compared with either the triple
mutant, containing Thr-356/Ser-807/Ser-811 only ( , p < 0.005), or with wild type ( , p < 0.0005). This
mutant was also capable of decreasing the level of cycling cells to a
level significantly below that observed in serum-deprived cells with
eGFP-transfected cells in the absence of serum ( , <0.005).
|
|
The effect of co-expression of cyclin A and pRb (wild type or mutants)
on cell cycle progression is shown in Fig. 5B. Previous studies have demonstrated that pRb, ectopically co-expressed with cyclin A, is fully phosphorylated and inactivated (28). Consistent with
these previous findings, our results revealed that there is no
significant difference in the size of the cycling cell population in
cells expressing transfected cyclin A, with (Fig. 5B,
lane 4) or without (Fig. 5B,
lane 3) co-expressed wild type pRb. However, when
pRb was mutated at either the m89 region or at the cdk sites Ser-807/Ser-811, the size of the cycling cell population was
significantly decreased and to the same extent (Fig. 5B,
lanes 5 and 6).
As the four cdk sites regulate the conformation of pRb independently,
we tested the effect of mutating additional cdk sites on the
progression of the cell cycle. The data showed that the addition of a
mutation at Thr-356 to mutations at Ser-807/Ser-811 caused a relatively
small decrease in the number of cycling cells when compared with the
Ser-807/Ser-811 mutation alone (Fig. 5B, lane
7). The most dramatic decrease in the cycling population occurred in the cells transfected with
peGFP/CycA/pRbmT356/S807/S811/T821 expressing pRb mutated at all four
sites (Fig. 5B, lane 8). Although co-expressed with cyclin A, this form of pRb reduced the cycling population of the transfected cells to a level (Fig. 5B,
lane 8) even lower than that observed in
serum-deprived peGFP (no cyclin A)-transfected cells (Fig.
5B, lane 2).
 |
DISCUSSION |
The Phosphorylation Status of Four cdk Sites Determines the
Conformation of pRb--
The data presented here describe the
identification of four cdk sites, the phosphorylation status of which
determines the conformation of pRb. Several conclusions can be drawn
from these data, summarized in Fig. 3B. First, the
phosphorylation status of each of the four cdk sites (Thr-356,
Ser-807/Ser-811, Thr-821) can independently control the conformation of
pRb. Second, the conformational changes apparently affect different
domains of pRb, such that mutation of more than one cdk site leads to
further conformational changes resolvable by SDS-PAGE. Third, the
conformation of pRb appears to be determined mainly by these four
sites, as the mutation of other cdk sites has little effect. Finally,
the five forms of pRb extracted from randomly growing cell cultures can
be attributed to different conformations created by the differential phosphorylation of these four sites. The observed greater intensity of
form 4 on SDS-PAGE can be explained simply by the observation that the
conformation brought about by an unphosphorylated Thr-356 and/or
Thr-821 cannot be resolved by one-dimensional SDS-PAGE. One of these
four sites, Thr-356, was previously reported to be inconsequential to
the determination of the conformation of pRb (12). The data presented
here demonstrated a drastic effect of the mutation of this site, alone
or in combination with the others, on the conformation of pRb. Thus,
the conformation of pRb is regulated by cdk sites both at the N
terminus and the C terminus.
Functional Significance of the Four cdk Sites and the Conformation
of pRb at the Molecular Level--
pRb is a binding target for many
proteins. Different domains are required for binding to different
proteins. A number of proteins, such as MCM7 and Ap-48 (29, 30) bind to
the N terminus of pRb, whereas RBP-1, RBP-2 (31), PU.1 (32), cyclin D
(33, 34), E2F-1 (35-37), histone deacetylase (38), and others bind to
the pocket region, while c-Abl binds exclusively to the c-terminus (11). The fact that some of these binding proteins, such as c-Abl and
E2F-1 (11) and HPV E7 and E2F-1 (39), can bind to pRb independently and
simultaneously further confirms that each of these proteins recognizes
a non-overlapping domain of pRb. Our data, which suggest that each of
the four cdk sites can independently regulate the conformation of a
particular domain, suggest that the phosphorylation status of these
sites may affect the binding of pRb to a particular binding protein.
Indeed, previous data suggest that certain proteins always recognize
particular forms of pRb. The phosphorylation status of Thr-821 and
Thr-826 affects the interaction of pRb with the
LXCXE motif, whereas that of Ser-807/Ser-811 affects pRb interaction with c-Abl. How the phosphorylation status of
Thr-356 may affect the interaction between pRb and pRb-binding proteins
that bind to its N terminus remains to be determined.
In addition, it is entirely possible that there are other functionally
important conformational changes in pRb brought about by the
phosphorylation of other cdk sites which are too subtle to be detected
by one-dimensional SDS-PAGE analysis. In fact, it was recently reported
that, although the phosphorylation status of Ser-795 does not
noticeably affect the conformation of pRb, it is nonetheless
functionally important (27). On the other hand, our data on the
influence of the mutation at the cdk sites on cell cycle progression
does support the contention of an important role of the phosphorylation
status-related conformational change in the proper functioning of pRb.
Functional Significance of the Critical cdk Sites and the
Conformation of pRb at the Cellular Level--
There are three lines
of evidence that support an important role for the four cdk sites in
proper pRb function. First of all, the active form of pRb from
TGF
-growth-arrested cells contains unphosphorylated Ser-807/Ser-811
sites. TGF
treatment results in down-regulation of cdk activity and
up-regulation of cdk inhibitors p27 and p15 (40-44), and the
phosphorylation of most cdk sites in pRb is down-regulated. The Ser-608
site, for example, is inefficiently phosphorylated under this condition
(26). Interestingly, five pRb cdk sites, at Thr-5, Ser-252, Ser-373,
Ser-788, and Ser-795, are also potential kinase sites for
mitogen-activated protein kinase. Recent studies suggest that some
mitogen-activated protein kinase-related kinases are activated when
TGF
is used as an inhibitor of epithelial cell growth (45). The
inability of TGF
treatment to down-regulate the phosphorylation of
these sites could be due to this overlap of kinase specificity. The
phosphorylation of Ser-807/Ser-811, on the other hand, appears to be
most severely affected, as shown by the complete absence of detectable
phosphopeptides when the amount of protein used for the analysis was
adjusted such that phosphopeptides for other sites are detected at high level. As there are a number of cdk sites that are substrates for
cyclin D1/cdk4 and cdk6, the more severe inhibition of phosphorylation at Ser-807/Ser-811 perhaps suggests these two sites are less accessible to the lowered level of cyclin D/cdk4/6. Our previous data showed that
the progression of the cells into S is accompanied by the heavy
phosphorylation of these two sites (14).
A second line of evidence that supports an important role for the four
cdk sites in proper pRb function comes from the observation that cell
cycle progression is more effectively blocked by ectopically expressed
cdk site pRb mutants than by wild type pRb. Although the finding that
serum deprivation can block cell cycle progression in pRb-negative
cells (Fig. 5B, lane 2) suggests that
active pRb is not the only critical factor for transducing a growth
arrest signal, our data indicate that expression of cyclin A in these cells is quite effective in reversing the serum deprivation effect. In
addition, this effect of cyclin A can be most successfully counteracted
by pRb in which the Ser-807/Ser-811 cdk sites were mutated. A similar
effect of the expression of cyclin A on 807/811 was noted previously,
in Saos2 cells (11).
Of the four cdk sites, Thr-356 and Ser-807/Ser-811 are substrates
for cyclin D1/cdk4/cdk6 but not for cyclinA/cdk2 or cyclin E/cdk2,
whereas Thr-821 can be phosphorylated in vitro by either cyclin A/cdk2 or cyclin E/cdk2, but not by cyclin D/cdk4/6 (46). This
perhaps explains the observation that pRbmT356S807S811T821, in which
all four sites were resistant to phosphorylation, was more efficient
than pRbmT356S807S811 at blocking progression of the cell cycle in
cells overexpressing cyclin A. The fact that pRbmT356S807S811T821 was
the most efficient at blocking progression of the cell cycle, more so
than serum deprivation alone (Fig. 5B), perhaps reflects
residual phosphorylation of the cdk sites even in medium containing
0.1% serum. Finally, pRb mutated at the m89 region, which affects the
phosphorylation status of cdk sites Ser-807/Ser-811, resulted in a
block in cell cycle progression equivalent to that produced by pRb
mutated at Ser-807/Ser-811. Taken together, these data support the
claim that the Ser-807/Ser-811 cdk sites are critical in determining
the conformation and function of pRb.
The Significance of the m89 Motif--
Data presented here reveals
that mutation at m89 renders the Ser-807/Ser-811 sites inaccessible to
cdks and that this conformation is maintained at all phases of the cell
cycle. It is interesting to note that phosphorylation of
Ser-807/Ser-811, but not Thr-821 or Thr-356, is much more severely
inhibited in cells growth-arrested by treatment with TGF
. These
characteristics of the m89 mutant suggest that pRbm89 should have
superior growth suppressing activity over wild type under certain
growth conditions. This is indeed supported by the data presented here.
How the m89 region regulates the accessibility of Ser-807/Ser-811 is
far from clear, but the fact that the conformation is maintained at all
phases of the cell cycle suggests that m89 is engaged in an
intramolecular interaction. The mutation appears to affect,
selectively, the conformation of the immediate upstream region where
cdk site 807/811 is located. The phosphorylation status of these two
sites in turn affects the further upstream region where c-Abl binds. It
is of great interest that we have recently extended these studies and
discovered that the m89 region is part of an antigenically conserved
domain, p5, between pRb and the tumor suppressor protein p53. Moreover, mutation of the p5 region in p53 also induces a conformational change
in the upstream cdk site Ser-315, such that it becomes inaccessible for
cdk phosphorylation (47). The phosphorylation status of this serine, in
turn, controls the ability of p53 to bind to its target DNA sequence
(48). It is possible that the presence of this regulatory domain in
both tumor suppressor proteins may afford a coordinated regulation of
their activities under certain growth conditions.
 |
ACKNOWLEDGEMENTS |
We thank David O. Morgan for the gift of the
cyclin A and cdk2 baculoviruses. Technical assistance was provided by
Xu-Xian Zhang, and Fu-Hui Zhang.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
(NIH) Grant RO1-CA44754 (to Y.K.F.), grants from the T. J. Martell Foundation for Leukemia, Cancer, and Aids Research and the Clayton Foundation for Research (to A. L. M.), and NIH Grant RO1-HL
54850 (to K. 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.
Current address: Dept. of Surgical Research, Childrens Hospital
Los Angeles, and Dept. of Surgery, University of Southern California,
School of Medicine, Los Angeles, CA 90027.
§
To whom correspondence should be addressed: Childrens Hospital Los
Angeles, Smith Research Tower, MS94, 4650 Sunset Blvd., Los Angeles, CA
90027. Tel.: 323-669-2595; Fax: 323-666-5975; E-mail:
yfung{at}chla.usc.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
PAGE, polyacrylamide
gel electrophoresis;
cdk, cyclin-dependent kinase;
TGF
, transforming growth factor-
;
GFP, green fluorescent protein;
eGFP, enhanced green fluorescent protein;
FACS, fluorescence-activated cell
sorting.
 |
REFERENCES |
-
Tung, J. S.,
and Knight, C. A.
(1971)
Biochem. Biophys. Res. Commun.
42,
1117-1121[Medline]
[Order article via Infotrieve]
-
Ghabrial, S. A.,
and Lister, R. M.
(1973)
Virology
51,
485-488[Medline]
[Order article via Infotrieve]
-
Mihara, K.,
Cao, X.-R.,
Yen, A,
Chandler, S.,
Driscoll, B.,
Murphree, A. L.,
T'Ang, A,
and Fung, Y.-K. T.
(1989)
Science
246,
1300-1303[Medline]
[Order article via Infotrieve]
-
Furukawa, Y.,
DeCaprio, J.,
Freedman, A.,
Kanakura, Y.,
Nakamura, M.,
Ernst, T. J.,
Livingston, D. M.,
and Griffin, J. D.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
2770-2774[Abstract]
-
Futreal, P. A.,
and Barrett, J. C.
(1991)
Oncogene
6,
1109-1134[Medline]
[Order article via Infotrieve]
-
Ludlow, J. W.,
Shon, J.,
Pipas, J. M.,
Livingston, D. M.,
and DeCaprio, J. A.
(1990)
Cell
60,
387-396[Medline]
[Order article via Infotrieve]
-
Hinds, P. W.,
Mittnacht, S.,
Dulic, V.,
Arnold, A.,
Reed, S. I.,
and Weinberg, R. A.
(1992)
Cell
70,
993-1006[Medline]
[Order article via Infotrieve]
-
Ewen, M. E.,
Sluss, H. K.,
Sherr, C. J.,
Matsushime, H.,
Kato, J.,
and Livingston, D. M.
(1993)
Cell
73,
487-497[Medline]
[Order article via Infotrieve]
-
Wang, J. Y. J.,
Knudsen, E. S.,
and Welch, P. J.
(1994)
Adv. Cancer Res.
64,
25-85[Medline]
[Order article via Infotrieve]
-
Mittnacht, S.,
and Weinberg, R. A.
(1991)
Cell
65,
381-393[Medline]
[Order article via Infotrieve]
-
Knudsen, E. S.,
and Wang, J. Y. J.
(1996)
J. Biol. Chem.
271,
8313-8320[Abstract/Free Full Text]
-
Hamel, P. A.,
Gill, R. M.,
Phillips, R. A.,
and Gallie, B. L.
(1992)
Oncogene
7,
693-701[Medline]
[Order article via Infotrieve]
-
Hamel, P. A.,
Cohen, B. L.,
Sorce, L. M.,
Gallie, B. L.,
and Phillips, R. A.
(1990)
Mol. Cel. Biol.
10,
6586-6595[Medline]
[Order article via Infotrieve]
-
Driscoll, B.,
Zhang, X.-X.,
T'Ang, A.,
Hu, Y.-H.,
Shi, X.-H.,
Zhang, F.-H.,
Wu, K.-J.,
Murphree, A. L.,
and Fung, Y.-K.
(1995)
Mol. Cell. Diff.
3,
361-375
-
DeCaprio, J. A.,
Furukawa, Y.,
Ajchenbaum, F.,
Griffin, J. D.,
and Livingston, D. M.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
1795-1798[Abstract]
-
Lin, B. T.-Y.,
Gruenwald, S.,
Morla, A. O.,
Lee, W. H.,
and Wang, J. Y. J.
(1991)
EMBO J.
10,
857-864[Abstract]
-
Mittnacht, S.,
Lees, J. A.,
Desai, D.,
Harlow, E.,
Morgan, D. O.,
and Weinberg, R. A.
(1994)
EMBO J.
13,
118-127[Abstract]
-
Kaye, F. J.,
Kratzke, R. A.,
Gerster, J. L.,
and Horowitz, J. M.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
6922-6926[Abstract]
-
Templeton, D. J.,
Park, S. H.,
Lanier, L.,
and Weinberg, R. A.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
3033-3037[Abstract]
-
Hansen, M. F.,
Morgan, R.,
Sandberg, A. A.,
and Cavanee, W. K.
(1990)
Cancer Genet. Cytogenet.
49,
15-23[Medline]
[Order article via Infotrieve]
-
Dryja, T. P.,
Rapaport, J.,
McGee, T. L.,
Nork, T. M.,
and Schwartz, T. L.
(1993)
Am. J. Hum. Genet.
52,
1122-1128[Medline]
[Order article via Infotrieve]
-
Fung, Y.-K. T.,
T'Ang, A.,
Murphree, A. L.,
Zhang, F. H.,
Qiu, W.-R.,
Wang, S.-W.,
Shi, X.- H.,
Lee, L.,
Driscoll, B.,
and Wu, K.-J.
(1993)
Oncogene
8,
2659-2672[Medline]
[Order article via Infotrieve]
-
Haas-Kogan, D. A.,
Kogan, S. G.,
Levi, D.,
Dazin, P.,
T'Ang, A.,
Fung, Y.-K. T.,
and Israel, M.
(1995)
EMBO J.
14,
461-472[Abstract]
-
Giannini, G.,
Di Marcotullio, L.,
Zazzaroni, F.,
Alesse, E.,
Zani, M.,
T'Ang, A.,
Sorentino, V.,
Screpanti, I.,
Frati, L.,
and Gulino, A.
(1997)
J. Biol. Chem.
272,
5313-5319[Abstract/Free Full Text]
-
Lees, J. A.,
Buchkovich, K. J.,
Marshak, D. R.,
Anderson, C. W.,
and Harlow, E.
(1991)
EMBO J.
10,
857-864[Abstract]
-
Zarkowska, T., U, S.,
Harlow, E.,
and Mittnacht, S.
(1997)
Oncogene
14,
249-254[CrossRef][Medline]
[Order article via Infotrieve]
-
Connel-Crowley, L.,
Harper, J. W.,
and Goodrich, D. W.
(1997)
Mol. Biol. Cell
8,
287-301[Abstract]
-
Dowdy, S. F.,
Hinds, P. W.,
Louie, K.,
Reed, S. I.,
Arnold, A.,
and Weinberg, R. A.
(1993)
Cell
73,
499-511[Medline]
[Order article via Infotrieve]
-
Sterner, J. M.,
Dew-Knight, S.,
Musahl, C.,
Kornbluth, S.,
and Horowitz, J. M.
(1998)
Mol. Cell Biol.
18,
2748-2757[Abstract/Free Full Text]
-
Huang, S.,
Lee, W.-H.,
and Lee, E. Y.-H. P.
(1991)
Nature
350,
160-162[CrossRef][Medline]
[Order article via Infotrieve]
-
Defeo-Jones,
Huang, P. S.,
Jones, R. E.,
Haskell, K. M.,
Vuocolo, G. A.,
Hanobik, M. G.,
Huber, M. E.,
and Oliff, A.
(1991)
Nature
352,
251-254[CrossRef][Medline]
[Order article via Infotrieve]
-
Hagemeier, C.,
Bannister, A. J.,
Cook, A.,
and Kouzarides.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
1580-1584[Abstract]
-
Ewen, M. E.,
Sluss, H. K.,
Sherr, C. J.,
Matsushime, H.,
Kato, J.,
and Livingston, D. M.
(1993)
Cell
73,
487-497[Medline]
[Order article via Infotrieve]
-
Kato, J.,
Matsushime, H.,
Hiebert, S. W.,
Ewen, M. E.,
and Sherr, C. J.
(1993)
Genes Dev.
7,
331-342[Abstract],
-
Bagchi, S.,
Weinmann, R.,
and Raychaudhuri, P.
(1991)
Cell
65,
1063-1072[Medline]
[Order article via Infotrieve]
-
Chellappan, S. P.,
Hiebert, S.,
Mudryj, M.,
Horowitz, J. M.,
and Nevins, J. R.
(1991)
Cell
65,
1053-1061[Medline]
[Order article via Infotrieve]
-
Bandara, L.,
Adamczewski, J. P.,
Hunt, T.,
and La Thangue, N. B.
(1991)
Nature
352,
249-251[CrossRef][Medline]
[Order article via Infotrieve]
-
Luo, R. X.,
Postigo, A. A.,
and Dean, D. C.
(1998)
Cell
92,
463-473[Medline]
[Order article via Infotrieve]
-
Wu, E. W.,
Clemens, K. E.,
Heck, D. V.,
and Munger, K.
(1993)
J. Virol.
67,
2402-2407[Abstract]
-
Ewen, M. E.,
Sluss, H. K.,
Whitehouse, L. L.,
and Livingston, D. M.
(1993)
Cell
74,
1009-1020[Medline]
[Order article via Infotrieve]
-
Polyak, K.,
Kato, J.-Y.,
Solomon, M. J.,
Sherr, C. J.,
Massague, J.,
Roberts, J. M.,
and Koff, A.
(1994)
Genes Dev.
8,
9-22[Abstract]
-
Slingerland, J. M.,
Hengst, L.,
Pan, C. H.,
Alexander, D.,
Stampfer, M. F.,
and Reed, S. I.
(1994)
Mol. Cell. Biol.
14,
3683-3694[Abstract]
-
Hannon, G. J.,
and Beach, D.
(1994)
Nature
371,
257-261[CrossRef][Medline]
[Order article via Infotrieve]
-
Koff, A.,
Ohtsuki, M.,
Polyak, K.,
Roberts, J. M.,
and Massague, J.
(1993)
Science
260,
536-539[Medline]
[Order article via Infotrieve]
-
Yan, Z.,
Winawer, S.,
and Friedman, E.
(1994)
J. Biol. Chem.
269,
13231-13237[Abstract/Free Full Text]
-
Zarkowska, T.,
and Mittnacht, S.
(1997)
J. Biol. Chem.
272,
12738-12746[Abstract/Free Full Text]
-
T'Ang, A.,
and Fung, Y. K. T.
(1995)
in
Challenges of Modern Medicine (Waxman, S., ed), Vol. 10, pp. 73-82, Ares-Serono Symposia, Rome
-
Wang, Y.,
and Prives, C.
(1995)
Nature
376,
88-91[CrossRef][Medline]
[Order article via Infotrieve]
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