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(Received for publication, September 28, 1994; and in revised form, December 19, 1994) From the
Many of the antimicrobial, immunomodulatory, and cell growth
regulatory activities of the interferons are mediated by
interferon-inducible proteins. One family of such murine proteins is
encoded by six or more adjacent and structurally related genes (gene
200 cluster). Two homologous human genes have also been reported. p202,
encoded by the Ifi202 gene in the gene 200 cluster, is a
52-kDa nuclear phosphoprotein. Constitutive overexpression of p202 in
transfected cells is growth-inhibitory. We report here that p202 binds
the cell growth regulatory retinoblastoma protein (pRb) in vitro and in vivo. The binding is due to direct interaction
between the two proteins. p202 has two nonoverlapping segments for
binding pRb, and pRb has two nonoverlapping segments (one of them
including the pocket region) for binding p202. The hypophosphorylated
form of pRb binds to p202. p202 is the first interferon-inducible
protein found to bind pRb.
The various activities of the interferons (IFNs) ( p202 is a 52-kDa
phosphoprotein. Its amino acid sequence contains potential sites for
phosphorylation by several kinases including
p34 The
105-kDa pRb is encoded by the retinoblastoma tumor suppressor gene and
is a negative growth regulator(14, 15, 16) .
The functioning of pRb is regulated by phosphorylation in a cell
cycle-dependent manner(17) . pRb is phosphorylated during the
late G IFNs can inhibit the growth of various cultured cells by prolonging
all phases of the cell cycle or by arresting the growth at the
G Here we report experiments demonstrating that
the IFN-inducible p202 protein binds pRb both in vitro and in vivo. The binding is a consequence of direct p202-pRb
interaction. p202 contains two nonoverlapping segments for binding pRb,
and pRb contains two nonoverlapping segments (one of them including the
pocket region) for binding p202. The hypophosphorylated
(growth-inhibitory) form of pRb is bound by p202.
To
obtain retinoblastoma templates for generating transcripts in vitro to be translated into truncated pRb segments, the pRb plasmid (in
pGEM3, Promega) was digested with appropriate restriction enzymes
(using NdeI for pRb-(1-479), Eco47III for
pRb-(1-254), and ClaI for pRb-(1-132)). The
plasmid encoding the GST-E1a fusion protein (pGST-E1a) was generated by
blunt-end ligating the EcoRI-SalI fragment from E1a
cDNA (from J. Germino) into the SmaI site of the GST vector.
The plasmid for the mammalian expression of p202 (pCMV-202) was
generated by ligating p202 cDNA into the BamHI site of the
pCMV vector (Invitrogen). The GST-human retinoblastoma segment fusion
proteins (GST-Rb-(379-928) and
GST-Rb-(379-928)(706C
Aliquots from the cell extracts (which had been diluted 2-fold with
extraction buffer) were incubated at 4 °C for 90 min with
glutathione-Sepharose beads loaded with the indicated GST fusion
protein. The beads were washed five times in extraction buffer
supplemented with 125 mM NaCl, and the bound proteins were
eluted as indicated above by boiling in sample buffer or at room
temperature with extraction buffer supplemented with 500 mM NaCl.
Wanting to test the effects of p202 overexpression, we have
transfected cultured AKR-2B cells with a plasmid encoding p202 cDNA,
driven by a strong enhancer and linked to a drug resistance (G418)
marker. Among the fewer than 30 drug-resistant clones obtained, the
level of p202 expression was only Since, as reported earlier(12) , the association of p202
with isolated nuclei seems to be based primarily on protein-protein
interaction, we wish to identify proteins that bind to p202 and,
possibly, mediate its growth-inhibitory action. A far Western assay
using labeled p202 as the probe for testing an extract from AKR-2B
cells revealed the binding of p202 to several proteins (data not
shown). The occurrence in the p202 sequence of LXCXE,
a motif known to mediate the binding of several proteins to the
negative growth regulator protein pRb (13) (Fig. 1),
prompted us to examine first whether p202 binds to pRb.
Figure 1:
Occurrence
of the pRb-binding motif LXCXE in p202. Shown is the
alignment of the motif in various pRb-binding proteins. HPV,
human papilloma virus.
Figure 2:
Binding of pRb to p202 in vitro. A: leftpanel, binding of pRb translated invitro to GST-202.
[
We proceeded by testing whether pRb
in cell extracts (which might be differently phosphorylated from that
translated in vitro) also binds to GST-202. As shown in Fig. 2B (leftpanel), both murine pRb
from NS1 or AKR-2B cells and human pRb from HeLa or U2OS cells bound to
GST-202, but not to GST. (The two faster migrating bands in lane2 correspond to degraded forms of pRb. These could be
detected because more pRb could be extracted from the NS1 cell line
than from the other lines tested.) No pRb was recovered on GST-202 from
an extract of cells of the human osteosarcoma line SAOS-2. This human
cell line does not express wild-type pRb; it expresses only a
cytoplasmic carboxyl-truncated version of pRb(32) . As
expected, p202 from an extract of AKR-2B cells bound to GST-Rb, but not
to GST (Fig. 2B, rightpanel, lanes5 and 6). p202 binding to GST-Rb was
detected, however, only in an extract from IFN-treated cells and not in
that from control cells (compare lanes6 and 4). This is due to the fact that the level of p202 is very low
in AKR-2B cells, not detected in our assay, unless the cells are
treated with IFN (lanes1 and 2). We also
used the far Western assay for testing whether labeled pRb can bind to
p202 specifically. The positive outcome of this test is shown in Fig. 2C. pRb bound to affinity-purified GST-202 (lane5), but not to GST or to a series of protein
size markers (lanes4 and 6). To test
whether the binding of p202 to pRb was due to direct interaction
between these two proteins, we used purified human recombinant pRb
(Canji, Inc.). This was retained on affinity-purified
GST-202-Sepharose, but not on GSTSepharose or the glutathione-Sepharose
matrix (Fig. 2D).
Figure 3:
Binding of the hypophosphorylated form of
pRb to GST-202 in vitro. Assay was by Western blotting.
Aliquots from an extract of growing AKR-2B cells were incubated with
glutathione-Sepharose beads loaded with equal amounts of GST (lane
2), GST-202 (lane 3), or GST-E1a (lane 4). After
washing the beads, the proteins were eluted and analyzed by SDS-PAGE
and Western blotting using a 1:1 mixture of antibodies to human pRb
(clones G3-245 and G99-549). The first of these two antibodies
recognizes all forms of pRb; the second antibody has a preference for
the hypophosphorylated form. An aliquot of the cell extract was
immunoprecipitated with anti-Rb antibodies (G3-245) and analyzed as a
control (lane 1). The arrows indicate differently
phosphorylated forms of pRb. For further details, see ``Materials
and Methods.''
Extracts were prepared from control
and IFN-treated cultures of untransfected and serum-starved and of
transfected and serum-starved AKR-2B cells. The extracts were used for
immunoprecipitation with monoclonal anti-Rb antibodies linked to
agarose beads. (These antibodies did not immunoprecipitate p202 that
was translated in a reticulocyte lysate, i.e. with no pRb
added (data not shown).) The immunoprecipitates from the cell extracts
were washed and analyzed by Western blotting using an anti-p202
antiserum. As a size marker, [
Figure 4:
Binding of pRb to p202 in vivo.
Assay was by coimmunoprecipitation and Western blotting. Extracts from
control (lane 1) or IFN-treated, serum-starved (lane
2) AKR-2B cells were immunoprecipitated using monoclonal anti-Rb
antibodies (Ab1-clone C36). The immunoprecipitated proteins were
analyzed by SDS-PAGE and Western blotting using anti-202 antiserum.
[
Figure 5:
Two nonoverlapping regions of pRb can
independently bind p202 in vitro. A, schematic
representation of pRb, its segments, and their abilities to bind p202. pRb refers to the murine retinoblastoma protein, and hpRb to the human retinoblastoma protein. The numbers not in
parentheses are the NH
Experiments
involving COOH-terminal deletion mutants of murine pRb confirmed and
extended these findings (Fig. 5C). Labeled
pRb-(1-922) (lane3), used as a control, and
the segments pRb-(1-479) (lane6) and
pRb-(1-254) (lane9) were retained on GST-202,
although not on GST (lanes2, 5, and 8). The short NH The
NH These results indicate that at
least two nonoverlapping regions of pRb can bind p202. One of them
includes the segment extending from amino acids 1 to 254. The second
one is located between amino acids 379 and 928. In this segment, the
pocket region is involved in the binding. A summary of the results
concerning the binding between pRb mutants and p202 is shown in Fig. 5A.
Figure 6:
Two nonoverlapping regions of p202 can
independently bind pRb in vitro. A, schematic
representation of p202 and its segments. The numbers in the structure
at the top indicate the amino acid residues at the ends of the
LXCXE pRb-binding motif. The lettersa and b are two types of partially conserved 200-amino acid
segments. The numbers at the left and right ends of the structures
indicate the NH
The results presented indicate that the IFN-inducible p202
protein binds the pRb protein both in vitro and in
vivo. p202 and pRb bind each other directly, as revealed by the
association of the purified recombinant proteins produced in bacteria.
p202 binds the hypophosphorylated form of pRb. At least two
nonoverlapping segments of p202 (p202-(58-291) and
p202-(295-445)) bound pRb. The latter of these contained the
pRb-binding motif LXCXE. p204, another protein that
is encoded by a gene (Ifi204) from the gene 200 cluster and
that is very similar to p202 in its COOH-terminal half(7) , did
not bind pRb under conditions in which p202 did. At least two
nonoverlapping segments of pRb (pRb-(1-254) and
pRb-(255-922)) bound p202. The second segment included the pocket
region. Deletions from the pocket region or an amino acid substitution
in it (at position 706) greatly diminished the binding to p202,
revealing the role of the pocket region in p202 binding. This is the
region to which the DNA tumor virus oncoproteins (adenovirus E1a,
simian virus 40 T, and human papilloma virus E7 proteins) (35, 36, 37, 38) as well as the
human cytomegalovirus IE2 protein also bind(39) . Furthermore,
these oncoproteins bind, at least preferentially, to the
hypophosphorylated form of pRb, the same form to which p202 also binds (40) . We used both murine and human pRb in our experiments.
The finding that human and murine pRb bound similarly to murine p202 is
not unexpected since there is 91% sequence identity between the two pRb
proteins(41) . The effect of p202 binding on pRb function
remains to be explored. It is conceivable, for example, that the
binding may impair the phosphorylation of pRb (IFN treatment was
reported to impair this process) (26) and/or the
phosphorylation-dependent release of pRb from its tight association
with the nucleus(42, 43) . The finding that p202 also
binds to the NH The binding of p202 to the pocket region of pRb might
account for the fact that p202 also binds to p107 (data not shown).
This protein is related in structure to pRb and is also involved in the
control of cell proliferation(46, 47) . The two
proteins are homologous in sequence in their carboxyl-terminal
two-thirds segments, including the pocket region, and the pocket
regions of the two proteins bind overlapping, although distinct, sets
of proteins. We detected an association in vivo of p202
with pRb only in an extract from IFN-treated cells. It remains to be
established whether this indicates that pRb interacts with p202 only if
the level of the latter has been increased by IFN treatment (a
15-20-fold increase in some cell lines)(12) . It might be
also be a consequence of the difficulty of the efficient extraction of
p202-pRb complexes from cell lysates at low salt concentration. This
difficulty may arise from the tight binding of hypophosphorylated pRb
and also of p202 to the nuclear fraction(12, 42) .
p202 has sequences that might be targets for phosphorylation by
p34 p202 is the first IFN-inducible protein
found to bind pRb, a protein with a crucial role in the negative
control of cell proliferation. At the same time, at least when
constitutively overexpressed, p202 impairs cell proliferation. These
facts warrant further studies on the possible role of the binding of
these two proteins in the growth-inhibitory activity of IFNs. In
considering this problem, it should be noted that p202 binds several
proteins in addition to pRb and p107 and that one of these proteins is
the transcription factor E2F (data not shown). This transcription
factor is involved in the G
Volume 270,
Number 11,
Issue of March 17, 1995 pp. 6134-6140
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)are mediated by IFN-inducible ``effector''
proteins(1, 2, 3, 4) . One family of
IFN-inducible proteins (including p202, p203, p204, and D3) is encoded
by six or more structurally related murine genes at the q21-q23
region of chromosome 1 (gene 200
cluster)(5, 6, 7, 8) . Two
homologous human genes (MNDA and IFI16) have also
been described (9, 10) . All the proteins encoded by
these genes share one or two homologous (partially conserved) 200-amino
acid-long segments. Three proteins (p202, p204, and MNDA) are
nuclear(9, 11, 12) .
(12) . The level of p202 is
increased in various cultured cells 15-20-fold after treatment
with IFN. After exposing cells to IFN, p202 accumulates first in the
cytoplasm and moves to the nucleus only after a delay of 
36 h. In
metaphase cells, p202 appears to be associated with chromatin. p202 is
not released from nuclei (isolated from IFN-treated cells) by DNase I
or low salt treatment. This suggests that the association of p202 with
the nucleus is not (or not only) due to binding to DNA, but may be a
consequence of binding to proteins. Constitutive overexpression of p202
in transfected cells is growth-inhibitory(12) . This activity
of p202, together with the occurrence in p202 of the amino acid
sequence LXCXE, also present in several (although not
all) proteins (13) binding the retinoblastoma protein (pRb),
prompted us to test for an interaction between p202 and pRb. and S phases and is dephosphorylated at the end of
mitosis(18, 19, 20) . Growth arrest caused by
deprivation of growth factors, high cell density, induction of
differentiation, or senescence is associated with the disappearance of
the hyperphosphorylated form of
pRb(19, 20, 21, 22) . The
hypophosphorylated form of pRb retains the cells in the
G
/G phase of the cell
cycle(18, 23) . Several cellular proteins interact
with pRb including the transcription factors E2F and DP1(24) . /G phase, e.g. in the case of
sensitive hematopoietic cells(25) . IFNs were also reported to
suppress phosphorylation of pRb in such hematopoietic
cells(26) .
Cell Lines, Growth Conditions, and Treatment with
IFN
Murine AKR-2B cloned embryo cells were cultured according to (11) . Murine NS1 myeloma and human J82 bladder carcinoma
cells, human U2OS osteosarcoma and SAOS-2 osteosarcoma cells (from
American Type Culture Collection), and human HeLa cervical carcinoma
cells (from G. Sen) were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum. When
indicated, the cells were treated with recombinant human
IFN-
/-(1-83) at 1000 units/ml
for 36 h (27) .Antibodies
Monoclonal anti-human pRb antibodies,
clone G3-245, and clone G99-549 were from Pharmingen. The monoclonal
anti-human pRb antibodies linked to agarose beads (Ab1-clone 36) were
from Oncogene Science Inc. A polyclonal rabbit anti-human pRb antiserum
(C-15) was from Santa Cruz Biotechnology Inc. All these antibodies
cross-react with murine pRb. The polyclonal rabbit anti-p202 antiserum
was described earlier(12) .Plasmids
The GST-202-(19-445) expression
plasmid (hereafter indicated as GST-202) was prepared in two steps. The HincII fragment from 202 cDNA (7) was inserted into
the SmaI site of the GST-1N vector (Pharmacia Biotech Inc.).
The plasmid obtained was cleaved with EcoRI (the resulting
short fragment was discarded), and the resulting long fragment was
ligated to the small EcoRI fragment that had been excised from
202 cDNA. GST-202-(58-291), GST-202-(255-445), and
GST-202-(295-445) were generated by ligating restriction
fragments from 202 cDNA (BspHI-EcoRI, BstEII-BamHI, and EcoRI fragments,
respectively) into the GST vector. GST-Rb-(237-922),
GST-Rb-(1-254), and GST-Rb-(255-922) were generated by
ligating restriction fragments from murine retinoblastoma cDNA (PvuII, HindIII-Eco47III, and Eco47III-HindIII fragments, respectively) into the SmaI site of the GST vector (Pharmacia Biotech Inc.).
F)) were from D. M. Livingston and W.
G. Kaelin, Jr.(28) .Expression of GST Fusion Proteins and Loading of
Glutathione-Sepharose Beads
The GST fusion proteins were
expressed in Escherichia coli DH5 and affinity-purified
as described by Kaelin et al.(28) . The beads were
loaded with 0.5 µg of the indicated GST fusion protein,
blocked in blocking buffer (0.25% gelatin, 50 mM KCl, 50
mM HEPES (pH 7.5)) at room temperature for 30 min, washed in
binding buffer (150 mM NaCl, 50 mM HEPES (pH 7.5),
0.1% Nonidet P-40), and used for affinity chromatography.Transcription and Translation in Vitro
Transcripts in vitro from 202 cDNA (in the pBluescript vector, Stratagene)
and murine pRb cDNA (in the pGEM3 vector) were translated in a rabbit
reticulocyte lysate (Promega) supplemented with
[
S]methionine(12) .Preparation of Extracts from Mammalian
Cells
Extracts from cultured cells were prepared in ice-cold
extraction buffer (50 mM HEPES (pH 7.5), 0.1% Nonidet P-40,
100 mM NaF, 0.2 mM orthovanadate, 100 µg/ml
leupeptin (Boehringer Mannheim), 1 mM phenylmethylsulfonyl
fluoride (Sigma)) supplemented with 250 mM NaCl. The extracts
were diluted 2-fold with (unsupplemented) extraction buffer for
affinity chromatography.Affinity Chromatography of Proteins Translated in Vitro
and Mammalian Cell Extracts on Glutathione-Sepharose Beads Loaded with
GST Fusion Proteins
Aliquots (10 µl) from the reaction
mixtures in which S-labeled proteins had been translated in vitro were incubated with washed glutathione-Sepharose
beads (loaded with the indicated GST fusion protein) in binding buffer
at room temperature for 30 min. The beads were washed five times in
binding buffer, and the bound proteins were released by boiling in
sample buffer (29) and subjected to SDS-PAGE and fluorography. Immunoblotting
Far Western blotting (30) was performed using [S]Met-labeled
murine pRb. Western blotting with anti-202 antiserum was performed
according to a published procedure (12) .Immunoprecipitation
Extracts from serum-starved
murine AKR-2B cells (control or treated with IFN) were incubated at 4
°C for 2 h with anti-Rb antibodies (Ab1-clone C36) that had been
conjugated to agarose beads. The beads were washed with extraction
buffer five times, and the bound proteins were released in extraction
buffer supplemented with 500 mM NaCl and subjected to SDS-PAGE
and Western blotting using the anti-202 antiserum.Expression of Murine pRb and p202 in Transfected
Cells
pRb-wt (from J. Sedivy) with murine retinoblastoma cDNA
inserted into a pMAMneo-Blue-derived expression vector containing the
cytomegalovirus enhancer (CLONTECH) and p202 cDNA in the form of
pCMV-202 were transfected into AKR-2B cells using a calcium
phosphate-based procedure(31) . Independent G418-resistant
clones were picked from the cultures transfected with pCMV-Rb,
pCMV-202, or pCMV vector. The three types of clones were pooled
separately, and the levels of pRb and p202 expressed were assayed by
Western blotting.
2-3-fold above the basal
level, and even these grew more slowly than control cells (data not
shown; see also (12) ). These findings, obtained repeatedly,
together with the fact that transfection of the vector only (with the
drug resistance marker) resulted in many more (over 3000) clones,
indicate that constitutive overexpression of p202 is growth-inhibitory.
Binding of p202 to pRb in Vitro
To facilitate the study,
we generated a GST-202 fusion protein (GST-202-(19-445)) in E. coli and affinity-purified it. Labeled murine pRb (which
had been translated in vitro) bound to GST-202, but not to GST (Fig. 2A, leftpanel). The
selectivity of the binding to GST-202 was indicated by the finding that
none of several labeled in vitro translated proteins, i.e. luciferase (Promega), three brome mosaic virus-encoded proteins
(of 109, 94, and 35 kDa) (Promega), or p204 (a protein with regions
similar in sequence to those in p202)(7) , bound to GST-202 or
to GST (data not shown). The binding of pRb to GST-202 reached the
maximal level within 5 min (data not shown). 15-20% of the input
pRb was bound even at 250 mM NaCl or in the presence of 5
mM EDTA. Labeled p202 (which had been translated in
vitro) bound to GST-Rb, but not to GST (Fig. 2A, rightpanel).
S]Met-labeled murine pRb was incubated with
glutathione-Sepharose beads loaded with either GST (lane2) or GST-202 (lane3). After washing
the beads, the bound proteins were eluted and analyzed by SDS-PAGE and
fluorography. An aliquot of [S]Met-labeled pRb (IVT-Rb) was run in lane1. The pRb band is
indicated by an arrow. Rightpanel, binding
of p202 translated in vitro to GST-Rb.
[S]Met-labeled p202 was incubated with
glutathione-Sepharose beads loaded with either GST (lane2) or GST-Rb (lane3). Processing was
as described for the leftpanel. An aliquot of
[S]Met-labeled p202 (IVT-202) was run
in lane1. The p202 band is indicated by an arrow. B: leftpanel, binding of
pRb in cell extracts to GST-202 assayed by Western blotting. Extracts
from the indicated murine (lanes 1-4) or human (lanes 5-10) lines were incubated with
glutathione-Sepharose beads loaded with either GST (lanes1, 3, 5, 7, and 9) or
GST-202 (lanes2, 4, 6, 8,
and 10). After washing the beads, the bound protein was
released by boiling in SDS sample buffer and was analyzed by SDS-PAGE
and Western blotting with the monoclonal anti-Rb antibody G3-245. The
pRb band is indicated by an arrow. Rightpanel, binding of p202 in cell extracts to GST-Rb assayed
by Western blotting. Extracts from growing AKR-2B cells without (lanes3 and 4) or after (lanes5 and 6) exposure to IFN were incubated with
glutathione-Sepharose beads loaded with GST (lanes3 and 5) or GST-Rb (lanes4 and 6). After washing the beads, the proteins were released by
boiling in SDS sample buffer and were analyzed by SDS-PAGE and Western
blotting with a polyclonal anti-p202 antiserum. Aliquots from extracts
of control and IFN-treated AKR-2B cells were also analyzed (lanes1 and 2, respectively). The p202 band is
indicated by an arrow. C: binding of pRb translated in vitro to GST-202 assayed by far Western blotting.
Affinity-purified GST-202 (2 µg) (lanes 2 and 5),
GST (5 µg) (lanes 1 and 4), and unstained protein
markers (PM) (myosin heavy chain, 200 kDa; phosphorylase b, 97 kDa; bovine serum albumin, 68 kDa; and ovalbumin, 43 kDa
(from Life Technologies, Inc.)) (lanes 3 and 6) were
subjected to SDS-PAGE, blotted to a membrane, and stained for proteins
with Ponceau S (lanes 1-3) or processed for far Western
blotting using labeled pRb translated in vitro (lanes
4-6). The p202 band is indicated by an arrow. D, direct binding of pRb to GST-202 assayed by Western
blotting. Purified recombinant pRb was incubated with
glutathione-Sepharose beads not loaded (indicated as GA; lane 1) or loaded with GST (lane 2) or GST-202 (lane 3). After washing the beads, the bound protein was
released and analyzed by SDS-PAGE and Western blotting using anti-Rb
antibodies. The pRb band is indicated by an arrow. For further
details, see ``Materials and
Methods.''
Binding of p202 to the Hypophosphorylated Form of pRb in
Vitro
In the experiments involving the binding of pRb from cell
extracts to p202, pRb appeared as a sharp protein band (Fig. 2B, left panel, lanes4, 6, and 10). The possibility that the
sharp pRb band may correspond to hypophosphorylated pRb prompted us to
examine whether p202 binds preferentially to the hypophosphorylated
form of pRb. We prepared an extract from growing (AKR-2B) cells to
assure that differently phosphorylated forms of pRb would be present.
We incubated aliquots from this extract with immobilized GST, GST-202,
and GST-E1a. The bound proteins were eluted, immunoprecipitated by
anti-pRb G3-245, and analyzed by SDS-PAGE and Western blotting with a
mixture of two monoclonal anti-human pRb antibodies (one of which
preferentially recognizes the hypophosphorylated form of pRb) (Fig. 3). As expected, no pRb was retained by GST. Only a fast
migrating band of pRb was retained on GST-202. The pRb retained on
GST-E1a consisted of a similarly fast moving band together with a
slower moving band. This is in line with earlier reports showing that
E1a binds both hypo- and hyperphosphorylated forms of pRb(33) .
Finally, the pRb bands from the cell extract that was used in the
experiment appeared to be similar to those recovered from GST-E1a.
These results indicate that from a mixture of differently
phosphorylated forms of pRb, p202 selectively, or at least
preferentially, binds to the fast migrating hypophosphorylated form.
Association of pRb with p202 in Vivo
Since p202
bound the hypophosphorylated form of pRb, we tested for the association
of p202 with pRb in vivo using an extract from cells that were
serum-starved to increase the proportion of pRb in the
hypophosphorylated form. Furthermore, since the level of p202 is very
low, barely detectable in AKR-2B cells not treated with IFN, we exposed
the cells to IFN for 36 h. Moreover, since the level of pRb in AKR-2B
cells is also very low, we transfected the cell line with an expression
plasmid (pRb-wt) (34) encoding wild-type murine pRb. This
provided us with AKR-2B cells expressing pRb at a 2-3-fold higher
level than in untransfected cells. These cells appeared morphologically
normal and grew similarly to cells that had been transfected with the
vector only (data not shown).S]Met-labeled p202
translated in vitro was run (Fig. 4, lane3). A strong p202-specific band was present in the
immunoprecipitate from the extract of IFN-treated, pRb-transfected
cells (lane2), suggesting that p202 and pRb may be
associated in vivo. The p202 band detected in the
immunoprecipitate from the extract of untransfected, IFN-treated cells
was weak presumably as a consequence of the low level of pRb in AKR-2B
cells (data not shown). No p202 was detected in the immunoprecipitates
from the extracts of transfected (or untransfected) control cells (lane1). In view of the very low level of p202 under
these conditions, this result was expected. We have not succeeded in
coprecipitating pRb from a cell extract using our polyclonal antiserum
to p202.
S]Met-labeled p202 translated in vitro (IVT-202) was run as a marker (lane 3). The p202
protein band is indicated by an arrow. For further details,
see ``Materials and Methods.''
Two Nonoverlapping Segments of pRb Bind to p202; One of
Them Includes the Pocket Region
We tested for interactions
between p202 and various truncated and/or mutated forms of murine and
human pRb (Fig. 5A). Labeled p202 was retained on two
nonoverlapping segments of murine pRb (linked to GST). One of them
extended in pRb from amino acids 1 to 254, and the second one from
amino acids 255 to 922 (Fig. 5B).
- and COOH-terminal residues of
the segment. (dl702-731) indicates that amino acids
702-731 were deleted (this is also indicated by the thickverticalline; (706,CF) indicates
that phenylalanine was substituted for cysteine at position 706. The letters A and B indicate two segments from the pocket
region. The extent of p202 binding is indicated in the right-most
column: -, no binding; + . . . ++++,
weakest to strongest binding. B, binding of p202 to segments
of pRb linked to GST. [S]Met-labeled murine p202
translated in vitro was incubated with glutathione-Sepharose
beads loaded with GST (lane 2), GST-Rb-(1-254) (lane
3), or GST-Rb-(255-922) (lane 4). After washing the
beads, the bound proteins were eluted and analyzed by SDS-PAGE and
fluorography. An aliquot of the [S]Met-labeled
p202 solution (IVT-202) was run in lane 1. The p202
band is indicated by an arrow. C, binding of murine
pRb segments with COOH-terminal truncations to GST-202.
[S]Met-labeled pRb (translated in
vitro) (lanes 1-3) or pRb segments with
COOH-terminal truncations (translated in vitro), i.e. pRb-(1-479) (lanes 4-6), pRb-(1-254) (lanes 7-9), and pRb-(1-132) (lanes
10-12), were incubated with glutathione-Sepharose beads
loaded with GST (lanes 2, 5, 8, and 11) or GST-202 (lanes 3, 6, 9, and 12). After washing, the beads were eluted, and the released
proteins were analyzed by SDS-PAGE and fluorography. As controls,
aliquots of the reaction mixture in which pRb or its segments with
COOH-terminal truncations had been translated in vitro were
run (IVT; lanes 1, 4, 7, and 10). The bands with the lowest mobilities (marked with open circles) correspond to the pRb segments indicated; the
faster moving bands may have arisen in consequence of translation
initiation at internal sites of the mRNAs and/or protein degradation. D, binding of p202 to GST-Rb with NH-terminal
truncation in the retinoblastoma moiety and an amino acid substitution
in its pocket region. [S]Met-labeled p202
translated in vitro was incubated with glutathione-Sepharose
beads not loaded (indicated as GA; lane 2) or loaded
with GST (lane 3), murine GST-Rb (lane 4), human
GST-Rb-(379-928)(706CF) (lane 5), or human
GST-Rb-(379-928) (lane 6). After washing the beads, the
proteins were eluted and analyzed by SDS-PAGE and a PhosphorImager. As
a control, an aliquot of the [S]Met-labeled p202
solution (IVT-202) was run in lane 1. The p202 band
is indicated by an arrow. E, binding of pRb with a
deletion in the pocket region to GST-E1a and GST-202. Extracts from
human HeLa cells carrying wild-type pRb (lanes 1-3) or
from human J82 cells carrying a pRb mutant with a deletion from amino
acids 702 to 731 (lanes 4-6) were incubated with
glutathione-Sepharose beads loaded with GST (lanes 1 and 4), GST-E1a (lanes 2 and 5), or GST-202 (lanes 3 and 6). After washing the beads, the
proteins were eluted and analyzed by SDS-PAGE and Western blotting with
a polyclonal anti-Rb antiserum. The pRb band is indicated by an arrowhead. For further details, see ``Materials and
Methods.''
-terminal segment
pRb-(1-132) was not retained on GST-202 (lane12) or on GST (lane11).-terminal truncation mutant of human pRb, i.e. pRb-(379-928), strongly retained labeled p202 (Fig. 5D). The same pRb segment (i.e. pRb-(379-928)), however, carrying an amino acid substitution
(Cys to Phe) at position 706 in the pocket region retained p202, but
very poorly (Fig. 5D). This result indicates that the
pocket region (extending from amino acids 379 to 792) is involved in
p202 binding. (In this experiment, a very faint band of p202 retained
on GST was detected. This might be a consequence of using a highly
sensitive detection device, a PhosphorImager (Molecular Dynamics,
Inc.). The conclusion concerning the involvement of the pRb pocket
region in p202 binding was further supported by the finding that the
natural human pRb mutant in J82 bladder carcinoma cells that harbor a
deletion from amino acids 697 to 731 in the pocket region (35) was retained by GST-202 to a much lesser extent than was
wild-type pRb from HeLa cells (Fig. 5e, compare lanes3 and 6). In accord with an earlier
report(35) , the binding of the mutant pRb from J82 to GST-E1a
was also much weaker than that of wild-type pRb (compare lanes2 and 5).Two Nonoverlapping Segments of p202 Bind pRb
We
generated p202 deletion mutants to identify regions involved in binding
pRb. Labeled pRb was bound to the following truncated GST-202 moieties:
GST-202-(58-291), GST-202-(255-445), and
GST-202-(295-445) (Fig. 6B, lanes
3-5), but not to GST (lane1). These
results suggest that p202 contains at least two nonoverlapping segments
that bind pRb: one between amino acids 58 and 291 and the second
between amino acids 295 and 445. The binding to p202-(58-291) and
also to the almost complete p202 (i.e. p202-(19-445))
was pronounced, but much weaker than to COOH-terminal
p202-(255-445) or p202-(295-445). It remains to be
established whether this is due to a masking in the almost complete
p202 of the activity of the stronger binding site in the COOH-terminal
segment. This COOH-terminal segment contains a pRb-binding motif,
LXCXE. The significance of this motif in the binding
of pRb to p202 remains to be tested.
- and COOH-terminal amino acid residues of
the p202 moieties. GST was linked to the NH terminus of
each of the lower four structures. B, binding of pRb to
segments of p202. Glutathione-Sepharose beads were loaded with GST (lane 1), GST-202-(19-445) (lane 2),
GST-202-(58-291) (lane 3), GST-202-(255-445) (lane 4), or GST-202-(295-445) (lane 5). The
loaded beads were incubated with [S]Met-labeled
pRb translated in vitro. After washing the beads, the proteins
were eluted and analyzed by SDS-PAGE and fluorography. An aliquot of
the [S]Met-labeled pRb preparation was also
analyzed (IVT-Rb; lane 6). The pRb band is indicated
by an arrow. For further details, see ``Materials and
Methods.''
-terminal region of pRb (reported to be
required for recognition by certain kinases (44) (and also for
the oligomerization of pRb)(45) ) is in line with these
possibilities.
, a cell cycle-dependent kinase(12) . Such
phosphorylation might control the ability of p202 to bind pRb in a cell
cycle-dependent manner. to S transition, and its
activity is controlled in part by pRb and
p107(48, 49) .
)
We thank C. Weissmann and H. Weber for recombinant
human IFN-/-(1-83); D. M.
Livingston and W. G. Kaelin, Jr. for the plasmids
pGST-Rb-(379-928) and pGST-Rb-(379-928)(706CF); J.
Germino for an E1a plasmid; J. Sedivy for the pRb-wt plasmid; G. Sen
for a HeLa strain; and J. Sedivy, M. Solomon, J. U. Gutterman, and B.
B. Aggarwal for valuable discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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