From the Molecular Oncogenesis Laboratory, Regina
Elena Cancer Institute, Rome 00158, Italy,
Department of
Biology, University of Tor Vergata, Rome 00133, Italy, the Departments
of ** Molecular Genetics and
Cell Biology,
The Weizmann Institute of Science, Rehovot 76100, Israel, and the
§§ Department of Medicine, Mount Sinai School of
Medicine, New York, New York 10029-6574
Received for publication, November 20, 2000, and in revised form, January 16, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Specific protein-protein interactions are
involved in a large number of cellular processes and are mainly
mediated by structurally and functionally defined domains. Here we
report that the nuclear phosphoprotein p73 can engage in a physical
association with the Yes-associated protein (YAP). This association
occurs under physiological conditions as shown by reciprocal
co-immunoprecipitation of complexes from lysates of P19 cells. The WW
domain of YAP and the PPPPY motif of p73 are directly involved in the
association. Furthermore, as required for ligands to group I WW
domains, the terminal tyrosine (Y) of the PPPPY
motif of p73 was shown to be essential for the association with YAP.
Unlike p73 The p53 tumor suppressor gene is the most frequent target for
genetic alterations in human cancer (1). The wild type
(WT)1 p53 protein is
apparently latent under normal conditions but becomes activated in
cells exposed to DNA damage as well as to other types of stress (2). As
a consequence of such stimuli, the level of p53 protein increases
suddenly and correlates a variety of antiproliferative effects,
including cell cycle arrest, apoptosis, and differentiation (3-5).
These biological effects are achieved mainly through the activation of
a plethora of specific target genes. For instance, p21waf1, a
cyclin-dependent kinase inhibitor, is mainly responsible
for p53-induced G1 arrest (6, 7). Furthermore, several
apoptotic proteins including Bax, are involved in the p53-induced
apoptosis (8, 9). Thus, the transcriptional activity of p53 is crucial for p53-induced cell cycle arrest as well as programmed cell death.
Two p53 homologues, p73 and p63, that share a remarkable homology in
DNA sequence as well as in protein structure have recently been
identified (11-14). As expected for p53-like proteins, p73 and p63 are
nuclear proteins that can bind to canonical p53 DNA binding sites and
can activate transcription from p53-responsive promoters in transiently
transfected cells. Furthermore, overproduction of p73 as well as p63
can induce apoptosis, growth arrest, and differentiation in p53+/+ and
p53 Viral oncoproteins such as large T antigen, E6, and E1Bp55, which are
known as inactivators of p53, are unable to promote the inactivation of
p73 (21-24). Similarly, Mdm2, a key regulator of p53 stability does
not induce degradation of p73 In an attempt to identify new proteins interacting with p73, we looked
at the ability of various SH3 as well as WW domains to bind to p73 Here we report a novel interaction between p73 and YAP. YAP was
originally identified as a protein binding to the SH3 domain of the Yes
proto-oncogene product that belongs to the Src family of
protein-tyrosine kinases. The interaction between p73 and YAP occurs
in vitro as well as under physiological conditions in the cell. It involves the WW domain of YAP and the PPPPY proline-rich region of p73 Library Construction--
Phage library construction
was according to Santi et al. (53) with minor modification.
The p73
The
The complexity of the libraries calculated as total independent clones
obtained after plating was 1 × 105 plaque-forming
units (the average length of the insert was 150 base pairs).
Affinity Selection--
Affinity selection was performed using
the glutathione S-transferase (GST)-YAP/WW bound to
glutathione-agarose beads (Sigma). Approximately 10 µg of protein
bound to resin were mixed with 400 µl of the p73
Cell Lines--
The H1299 cell line is derived from a human
large cell lung carcinoma. H1299 cells were maintained in RPMI medium,
supplemented with 10% fetal calf serum (Life Technologies, Inc.).
Before transfection, the culture medium was changed to Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum. H1299
cells stably overexpressing inducible WT p53 were produced as
previously reported (34, 55). Induction of WT p53 was achieved by the
addition of ponasterone A (2.5 µM/ml). P19 mouse
embryo-carcinoma cells were maintained in Plasmids and Transient Transfections--
Overexpression of p73
was achieved by transfection of pcDNA3-HA-p73 Coprecipitation and Western Blot Analysis--
H1299 cells were
transfected in 60-mm plates with 10 µg of total DNA and harvested at
36 h after the transfection. Cells were lysed in 900 µl of lysis
buffer (50 mM Tris (pH 8), 100 mM NaCl, 10%
glycerol, 1% Triton X-100, 1 mM EDTA, 100 mM
NaF, 1 mM MgCl2, 2 mM
phenylmethylsulfonyl fluoride, protease, and phosphatase inhibitors),
and the extracts were sonicated for 10 s and centrifuged at 14,000 rpm for 10 min to remove cell debris. Protein concentrations were determined by a colorimetric assay (Bio-Rad) assay. After preclearing for 1 h at 4 °C, immunoprecipitations were
performed by incubating 2 mg of whole-cell extract with 1.5 µg/sample
of anti-p73 mAb (Ab4) (Neomarkers Inc.) with rocking at 4 °C for 1 h. Immunocomplexes were precipitated with protein G-agarose beads (KPL). The immunoprecipitates were washed three times with 1 ml
of wash NET-gel buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.25% gelatin, 0.1%
Nonidet P-40). The excess liquid was aspirated, and 40 µl of 5×
sample buffer was added. Immunoprecipitates as well as 1% of each
extract were resolved by SDS-9% PAGE. Protein gels were transferred to
nitrocellulose membranes (Sartorius). For GFP-YAP detection, a
polyclonal anti-GFP antibody (Invitrogen) was used at 1:5000 dilution;
for p73 detection, an anti-p73 polyclonal serum was used at 1:3000. For
Bax detection, a polyclonal anti-Bax antibody (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA) was used at 1:500 dilution.
P19 cells were lysed as previously described. Aliquots of cell extracts
containing 5 mg of total proteins were immunoprecipitated with anti-YAP
affinity-purified polyclonal IgG (56), with anti-p73 polyclonal
serum, with anti-p53 mAb 421, or with a mixture of anti-p53 mAbs
DO1 and 1801. For p73 detection, an anti-p73 polyclonal serum was used
at 1:3000 dilution; for YAP detection, an anti-YAP polyclonal serum was
used at 1:3000 dilution.
Western blot analysis was performed with the aid of the enhanced
chemiluminescence Supersignal West Pico Stable Peroxidase Solution (Pierce).
Production of Recombinant Proteins--
GST fusion proteins
containing lengths p73 Pull-down Assays--
Pull-down assays were performed using 20 µg of immobilized purified GST fusion proteins or wild-type GST that
was incubated with 2.5 mg of total cellular proteins prepared from
H1299 cells transiently transfected with the following plasmids
encoding p73 Indirect Immunofluorescence--
H1299 cells transiently
transfected with the indicated combination of plasmids were first
incubated for 5 min at room temperature with a solution containing 10%
bovine serum albumin, 0.5% Tween 20 in 1× PBS (PAT solution) and then
fixed in 3.75% PBS-paraformaldeyde plus 0.1% Triton-100 for 10 min on
ice. After rehydration with PAT for 5 min, the cells were incubated in
50% PAT and then stained for 1 h with anti-p73 polyclonal serum
used at 1:100 dilution. Staining with the secondary antibody and with
Hoechst was performed as described before (57), followed by
visualization under a fluorescence microscope.
Luciferase Assays--
H1299 cells were transfected with
reporter plasmid together with the indicated expression plasmid
combinations. 36 h later, cells were rinsed with cold
phosphate-buffered saline, resuspended in cell lysis buffer (Promega),
and incubated for 10 min at room temperature (34). Insoluble material
was spun down, and luciferase activity was quantified using a
commercially available kit (Promega) with the aid of a TD-20E
luminometer (Turner).
p73
To explore the possibility that the association between p73 and YAP is
direct, we performed a pull-down assay incubating GST-YAP/WW or GST
fusion proteins with purified His-tagged p73
To test whether the association between p73
These results demonstrate that p73 The Association between p73 and YAP Occurs under
Physiological Conditions--
To verify whether the association of p73
and YAP occurs under physiological conditions, lysates of P19 cells
were immunoprecipitated with anti-YAP and as an internal control for
p53 family members, with a mixture of anti-human p53 mAbs DO1 and 1801. As shown in Fig. 2A,
endogenous p73 was co-immunoprecipitated by anti-YAP (lane
2) but not by anti-p53 antibodies (lane
3). In a reciprocal experiment, the same cell lysates were
first precleared with protein G-agarose beads and then subjected to
immunoprecipitation with an anti-p73 polyclonal serum or, as controls,
with anti-p53 mAb 421 or with a mixture of anti-p53 mAbs DO1 and 1801. Again the endogenous YAP was brought down by anti-p73 antibody but not
by anti-p53 mAbs DO1/1801 and 421 (Fig. 2B, lanes
1-4).
Taken together, these results suggest that the association between p73
and YAP occurs under physiological conditions. Furthermore, they show
that, at least under our experimental conditions, p53 does not
associate with YAP.
Differential Binding of YAP to the Various Members of the p53
Family--
The p73 Binding to and Tyrosine Phosphorylation of p73
First, we checked whether the p73
Second, by a similar approach, we proved that p73
Thus, we conclude that both the binding of p73 YAP Functions as a Transcriptional Co-activator of p73
To assess whether the association with YAP influences p73
To determine whether the enhancement of transcriptional activity
mediated by p73 A Single Point Mutation Tyr
In order to investigate the precise role of the PPPPY motif of p73
We next investigated whether binding to YAP is required for the
co-activation of p73
The reported results demonstrate that the integrity of the PPPPY motif
of p73 In the present study, we report that the WW domain of
YAP binds to a region of p73 immediately preceding the SAM domain.
Furthermore, we show that this same interaction mediates the
association between YAP and p73, under physiological conditions, as
shown in P19 cells by reciprocal co-immunoprecipitation experiments.
YAP is a ubiquitously expressed phosphoprotein interacting with the SH3
domain of the proto-oncogene protein c-Yes, a nonreceptor tyrosine
kinase that belongs to the Src family. Its WW domain preferentially
binds to ligands containing a PPXY motif and belongs to the
first class (class I) of the current classification of the diverse WW
domains (40, 41).
Specific protein-protein interactions are often mediated by
functionally and structurally defined families of small protein modules
(39, 62). We have shown that the association between p73 and YAP
involves the binding of the WW domain of YAP to a p73 region containing
a PPPPY motif. Unlike p73 Because of its modular structure and the absence of an
enzymatic activity, YAP can be classified as an adapter protein.
In vitro YAP was characterized as a ligand of cytoplasmic
kinases like c-Yes, c-Src, and Crk, but in vivo it is found
preferentially in the nucleus, suggesting that it might contribute to
transmit signals from the cytoplasm to the nuclear compartment (56,
61). For instance, c-Src is triggered by c-Fms/macrophage colony
stimulatory factor-1 receptor for macrophage colony stimulation
factor (63). p73-deficient mice show several defects including
inflammatory processes that might result from a deficiency in
appropriate differentiation along the white cell lineage. We and others
have observed that p73 protein is strongly induced upon macrophage
differentiation of HL-60
cells.3 Although these
considerations are, at the moment, rather speculative, they permit us
to logically connect extracellular signals to a nuclear output through
an interaction chain that includes YAP and p73. Thus, YAP might
integrate signals incoming from c-Src to activate p73. The similarity
between YAP and other signaling proteins that sense external signals
near the plasma membrane and transduce them to the nucleus is
reinforced by the recent observation that YAP localizes to the apical
plasma membrane in polarized airway epithelial cells by binding to the
second PDZ domain of EBP50 (64). YAP, in this respect, mimics the
behavior of Smad, Stat, or JAB1 proteins that, upon activation of the
tyrosine kinase activity associated with growth factor receptors or by integrin-mediated signals, translocate into the nucleus to form functional complexes with transcription factors (65, 66). These
considerations, taken together with the recent findings that YAP
associates with the transcription factor polyomavirus enhancer binding
protein 2 and the reported data showing the interaction with p73,
strongly indicate that the nucleus is the important site of YAP
activity (61). Thus, the PPPPY motif of p73, by binding to YAP, might
contribute to transmission of cytoplasmic signals to the nucleus. Here
we report that the binding to YAP enhances p73 A biological read-out for the complex formed by p73 and YAP is likely
to emerge from the study of muscle differentiation. YAP is expressed at
considerable levels in muscle cells, and p73 transcript is strongly
up-regulated along muscle differentiation (70).4 An additional facet of
YAP function in modulating transcriptional activity of p53 family
members was recently revealed by the characterization of a functional
complex between YAP and p53-binding protein 2 (71). It appears
that the interaction between p53-binding protein 2 and YAP renders
p53-binding protein 2 inactive in terms of complex formation with p53.
Thus YAP could influence the activity of the p53 family members either
by direct or indirect means.
Further analysis is required to verify whether differential
binding of YAP to the p53 family members might provide a molecular explanation to their functional divergence in signaling.
, p73
, and p63
, which bind to YAP, the
endogenous as well as exogenously expressed wild-type p53 (wt-p53) and
the p73
isoform do not interact with YAP. Indeed, we documented that
YAP interacts only with those members of the p53 family that have a
well conserved PPXY motif, a target sequence for WW
domains. Overexpression of YAP causes an increase of p73
transcriptional activity. Differential interaction of YAP with members
of the p53 family may provide a molecular explanation for their
functional divergence in signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
tumor cells (10, 14, 15, 16). In contrast to p53, p73 and p63
are alternatively spliced, giving rise to a family of different
isoforms whose individual physiological functions are still unknown
(10, 14, 17-20).
, although it binds to it and
interferes with its transcriptional activity (25-29). Taken together,
these results suggest that the mechanisms underlying p73
stability
are distinct from those known to regulate p53 stability. Recently, a
growing number of proteins have been identified as p73 binding partners
(30, 31). It was originally reported that human tumor-derived p53
mutants (m-p53) bind to p73 and markedly decrease its ability to
transactivate target genes as well as to induce apoptosis (32). It was
subsequently reported that m-p53 can associate in vivo with
all of the isoforms of p73 (33, 34). Different types of DNA damage
cause different p73-mediated responses. Unlike UV irradiation,
cisplatin as well as
-radiation cause stabilization and tyrosine
phosphorylation of p73 (35-37). These post-translational modifications
of p73 occur through the physical interaction with kinase-active c-Abl
and result in the enhancement of the apoptotic activity of p73
(35-37). The proline-rich region of p73
and p73
mediates the
association through the interaction with the SH3 domain of c-Abl (36,
37).
in pull-down assays. Although structurally distinct, SH3 and WW domains
are functionally related due to their ability to bind to proline-rich
ligands. WW domains are small protein modules composed of 38-40 amino
acids and characterized by two conserved tryptophan residues that are
20 residues apart (38-40). WW domains can be grouped into four classes
according to their ligand binding preference (39, 40). Class I includes
WW domains binding to the core sequence PPXY
(41-44). Class II WW domains prefer ligands containing a stretch of
prolines interrupted by a leucine (45, 46). Class III includes WW
domains interacting with proline-rich sequence that contains arginines
or lysines (47-49). WW domains binding phosphoserine or
phosphothreonine followed by a proline residue are grouped in class IV
(50-52).
and p73
. In contrast to p73
and p73
and
p63
, p53 does not associate with YAP. The binding with YAP enhances transcriptional activity of p73
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
monkey gene was amplified from plasmid pCDNA3-HA p73
(15) using a primer annealing to region encoding the first 6 amino
acids of the protein (ATCGCGGATCCATGGCCCAGTCCACCACCAC), and a second
primer annealing to the region encoding the last 6 amino acids
(GACCGGATATCTCAGTGGATCTCGGCCTCCG). The PCR product was
electrophoresed on an agarose gel (1%) and purified using a NucleoSpin
column (Clontech). To generate first-strand cDNA copies, 1 µg of
this purified fragment was mixed with an SpeI-tagged random
primer (ACGCGGACTAGTN6), 5 µl of dNTP (25 mM each) and 7 µl of Klenow buffer (New England Biolabs)
in a final volume of 70 µl, boiled for 5 min, and immediately chilled
on ice. Afterward, 10 units of Klenow (New England Biolabs) were added
and incubated for 2 h at 37 °C. This mixture was purified
through a Quiaquick column (Qiagen) and used to generate the
second-strand cDNA with a NotI tagged random primer
(TCGGCGGCCGCN6) in the same way as described above.
The resulting double-stranded inserts were amplified using
SpeI (CTGCTGACGCGGACTAGT) and NotI primers
(TGGATCTCGGCGGCCGC) in order to generate fragments in the three
possible reading frames. Products were purified and digested with
SpeI and NotI (Promega).
display 3 vector was digested by SpeI and
NotI, purified, and concentrated by isopropanol
precipitation. The ligation of the vector (2 µg) and the inserts (15 ng) was performed by adding 2000 units of T4 DNA ligase (New England
Biolabs) and incubating overnight at 16 °C. The ligation mixture was
in vitro packaged by using a
packaging kit, Gigapack
(Stratagene), and plated by infection of BB4 cells onto 10 plates
(15-cm diameter). Phage elution was achieved with the standard method
(54).
phage library
(1 × 1010 plaque-forming units) with 1% bovine serum
albumin and incubated overnight at 4 °C. The resin was washed five
times with 1 ml of TBST and then once with 1 ml of
buffer and
resuspended in 100 µl of the same buffer. Selected phages were
recovered by adding 200 µl of BB4 cells in 10 mM
MgSO4 at A600 = 2 and incubating for
20 min at 37 °C. Infected cells were collected and plated onto one
plate (15 cm). After overnight incubation at 37 °C, plaque-forming phage were collected with the standard method (54). The phages that
specifically bound to YAP/WW were evidenced with an immunoscreening assay performed as described by Zucconi et
al.2
plaques that were found to be positive in a plaque-screening assay
were isolated and eluted into 50 µl of
buffer, and 1 µl of the
suspension was PCR-amplified for sequencing of the DNA insert.
-minimal essential
medium (Life Technologies, Inc.) without ribonucleotides and
deoxyribonucleotides supplemented with 7.5% newborn calf serum (Life
Technologies, Inc.) and 2.5% fetal calf serum.
(kindly provided
by Dr. W. Kaelin) and pcDNA3-HA-p73
(kindly provided by
Dr. G Melino). pCDNA3-HA-p73
,Y99-F,
pCDNA3-HA-p73
,Y121-F,
pCDNA3-HA-3
, Y99/121-F,
pcDNA3-HA-p73
,P338A, and pcDNA3-HA-p73
,Y487P were
obtained by site-directed PCR mutagenesis followed by subcloning into
pCDNA3-HA vector. Sequences of the oligonucleotides and primers are
available on request. Overexpression of GFP-YAP and GFP was achieved by transfection of the plasmids pEGFP-YAP and pEGFP-C2. The parental vectors pCMVneo or pEGFP were used to keep the amount of the
transfected DNA constant among samples. Transient transfections were
done by the calcium phosphate method in the presence of BES (Sigma). The precipitates were left for 12 h, after which the medium was changed again to RPMI plus 10% fetal calf serum. The cells were harvested 36 h posttransfection.
, p73
, p63
, and p53 were obtained
by PCR amplification of the appropriate fragment from monkey p73, mouse
p63, and mouse p53 followed by cloning in the pGEX-2KT expression
vector in frame with the GST moiety. The p73
mutant Y487F was
obtained by site-directed PCR mutagenesis followed by cloning in the
pGEX-6P1 (Amersham Pharmacia Biotech). YAP/WW was obtained by PCR
amplification followed by cloning in the pGEX-2KT. Sequences of the
oligonucleotides and primers used are available on request.
Purification of the GST fusion proteins by glutathione-agarose beads
(Sigma) was performed following standard procedures (34). H6-p73
was
obtained by PCR amplification followed by cloning in the pQE30 vector
(Qiagen). Purification and elution of H6-p73
protein were performed
following standard procedures.
-HA, p73
-HA (Y99F), p73
-HA (Y121F),
p73
-HA (Y99F/Y121F), p73
-HA (P338A), p73
-HA, GFP-YAP,
or GFP. The lysates were first precleared with glutathione-agarose
beads and then incubated for 2 h at 4 °C. Following three
consecutive washes with HNTG buffer (20 mM Hepes (pH 7.5),
150 mM NaCl, 0.1% Triton X-100, 10% glycerol), excess
liquid was aspirated, and 40 µl of 5× sample buffer was added.
Immunoprecipitates and 1% of each extract were resolved by SDS-9%
PAGE. The immunoblots were probed with anti-p73, anti-GFP, or anti-YAP
polyclonal serum. Detection was performed with the aid of the enhanced
chemiluminescence Supersignal West Pico Stable Peroxidase Solution. (Pierce).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Associates in Vitro and in Vivo with YAP--
In a search
for new p73-binding proteins, we checked whether various SH3 as well as
WW domains associate with p73
in pull-down assays. To this end,
H1299 cells were transiently transfected either with an empty vector
(H1299) or with a vector encoding a hemagglutinin (HA)-tagged version
of p73
(H-p73
) (15). Equal amounts of protein lysates were first
precleared with glutathione-agarose beads and then incubated with GST
fusions of SH3 or WW domains (listed in Table
I). The pulled down proteins were
separated by gel electrophoresis on an SDS-9% PAGE, transferred to
nitrocellulose membranes, and probed with anti-p73 polyclonal serum
(34). As shown in Fig. 1A,
p73
was pulled down by human YAP WW domain (lanes
5 and 6) only when incubated with protein lysates
of H-p73
cells. No p73
protein was pulled down when lysates of
H1299 cells transfected with an empty vector were incubated with
GST-YAP/WW or with GST alone (lanes 2 and
3). 1% of unprocessed cell lysates derived from either
H1299 or H-p73
cells were concomitantly run on the gel
(lanes 1 and 4).
Interactions between SH3 and WW domain-containing proteins and
p73.
was tested as
reported in Fig. 1a.
View larger version (17K):
[in a new window]
Fig. 1.
p73 associates
in vitro and in vivo with YAP.
A, H1299 cells were transiently transfected either with a
vector encoding p73
-HA or with an empty vector. Cell lysates (2 mg)
were first precleared with glutathione-agarose beads and then incubated
with the indicated GST fusion proteins for 2 h at 4 °C.
Specifically bound p73
was detected by immunoblotting with anti-p73
polyclonal serum. Lanes 1 and 4 contain aliquots of unprocessed lysates (100 µg/lane), loaded
directly on the gel. B, bacterial purified H6-p73
protein
was incubated either with GST-YAP/WW (lane 3) or
with GST alone (lane 2) and
transferred to nitrocellulose membrane. The blot was probed with
anti-p73 polyclonal serum (upper panel).
The same blot was reprobed with anti-GST polyclonal serum
(lower panel). C, p73
as well as
GFP-YAP or GFP alone were overexpressed in H1299 cells by transient
transfection. Cell extracts were precleared with protein G-agarose and
followed by immunoprecipitation (IP) with anti-p73 antibody.
Immunoprecipitates were subjected to immunoblot with anti-GFP
polyclonal serum (upper panel). The same blot was
reprobed with anti-p73 polyclonal serum (middle upper
panel). Aliquots of total cell extracts from unprocessed
cells (100 µg/lane) were directly subjected to immunoblot analysis
(lower panels). D, subcellular
localization of GFP-YAP and p73
is shown. Cells were stained with
Hoechst to visualize nuclei and anti-p73 polyclonal serum to visualize
p73. Protein molecular sizes markers are indicated on the
left.
(H6-p73
) protein expressed in bacteria. As shown in Fig. 1B, purified
H6-p73
protein efficiently binds to GST-YAP/WW
(upper panel, lanes 2 and
3). In order to show the presence of GST fusion proteins, the lower part of the same blot was probed with anti-GST polyclonal serum (Fig. 1B, lower panel).
and YAP would occur in a
cell, H1299 cells were transiently transfected with a combination of
vectors encoding p73
-HA and a GFP-tagged version of human YAP. Cell
lysates derived from the above mentioned cells were first precleared
with protein G-agarose beads and then subjected to immunoprecipitation
with a mixture of anti-p73 polyclonal sera (34). The immunoprecipitates
were separated on SDS-9% PAGE, transferred to nitrocellulose
membranes, and probed with anti-GFP polyclonal serum. As shown in Fig.
1C, a coprecipitated GFP-tagged YAP was detected only when
both p73
and YAP were co-expressed (upper
panel, lane 3). For control, the same
blot was reprobed with anti-p73 polyclonal serum (Fig. 1C,
middle upper panel, lanes 2 and 3). The protein levels of GFP-YAP
(lane 3) and GFP alone (lane
2) are shown in the lower panels of
Fig. 1C. Immunostaining analysis revealed a predominant
nuclear localization for both p73
and GFP-YAP when transiently
co-expressed in H1299 cells (Fig. 1D).
associates with YAP in
vitro and in vivo and that this association might occur directly.
View larger version (11K):
[in a new window]
Fig. 2.
The association between p73 and YAP occurs
under physiological conditions. P19 mouse embryo-carcinoma cells
were extracted and subjected to reciprocal co-immunoprecipitation as
described under "Experimental Procedures." A,
lanes 2 and 3 represent
immunoprecipitates (IP) corresponding to 5 mg of total cell
protein performed with indicated antibodies. Lane
1 contains an aliquot (100 µg/lane) of unprocessed extract
of the above indicated cells applied directly on the gel. The blot was
probed with anti-p73 polyclonal serum. B, an identical
amount of cell lysate employed in A was immunoprecipitated
with the indicated antibodies. An aliquot (50 µg/ml) of unprocessed
extract was run in lane 1. The blot was probed
with anti-YAP polyclonal serum.
sequence contains two peptides whose sequences
match the PPXY binding consensus for the YAP-WW domain (40,
41). The first peptide, 403QPPSYG409 is
at the carboxy-side of the oligomerization domain, while the second, 482PPPPY488, immediately
precedes the SAM domain. To distinguish between the two candidate
target sites, we have generated fragments of the p73 coding sequence
and expressed them as part of the gene for the lambda D capsid protein
(see "Experimental Procedures"). The resulting chimeric phages
displayed different fragments of the p73 protein on the capsid. By
panning this repertoire with the YAP-WW fused to GST, we have selected
the clones that display a peptide fragment that binds to YAP. Only
those phage-displayed peptides that only contained the
482PPPPY488 were selected, suggesting that this
peptide could be sufficient for the formation of the complex with the
WW domain of YAP (Fig. 3A).
Since this consensus is conserved in p73
, p73
, and p63
but is
not present in p53 and p73
(Fig. 3B), this result would imply that YAP associates selectively with the different protein products of the p53 gene family. To investigate this possibility, H1299
cells were transiently transfected either with a vector encoding a
GFP-tagged version of human YAP or with an empty vector. Cell lysates
were first precleared with glutathione-agarose beads and then incubated
with p73
, p73
, p63
, and p53 GST fusion proteins as well as
with GST alone. After separation on SDS-9% PAGE, the pulled down
proteins were transferred to nitrocellulose membranes and probed with
anti-GFP polyclonal serum. In agreement with the results reported
above, YAP binds to p73
, p73
, and p63
but not p53 (Figs.
4A and
5, A and B). The
absence of the association between p53 and YAP was further investigated
by pull-down assays in P19 as well as in H1299 cells stably
overexpressing ponasterone-induced human WT p53 (H-p53) (55). As
reported in Fig. 5, C and D, incubation of cell
lysates of either P19 or H-p53 cells with GST-YAP/WW or with GST alone
did not reveal any specific binding with p53. Aliquots of unprocessed
cell lysates (100 µg/lane) derived from both types of cells were
concomitantly run to show endogenous p53 in P19 (Fig. 5C,
lane 1) and exogenous p53 in H1299 cells (Fig.
5D, lane 4). To verify whether YAP
would bind to the differentially spliced variants of p73, H1299 cells
were transiently transfected with vectors encoding p73
or p73
or
with empty vector. Precleared cell lysates were incubated with
GST-YAP/WW or with GST alone. As shown in Fig. 4B, in
contrast to p73
, p73
does not associate with YAP. Coomassie Blue
staining of replica gels showed equal amounts of recombinants proteins
(Figs. 4B and 5D, lower
panels). Taken together, these data suggest that binding to
the PPPPY motif governs the specificity in the association between YAP
and the members of the p53 family.
View larger version (30K):
[in a new window]
Fig. 3.
The canonical PPPPY motif of p73 is directly
involved in the association with YAP. A, the two ligand
binding consensus sequences for YAP are depicted along the
schematic representation of p73 modular structure. The clones selected
by phage display analysis performed as reported under "Experimental
Procedures," and the PPPPY motif of each clone is marked in
gray. B, schematic representation of YAP
ligand binding consensus on the sequences of p73 , p73
, p73
,
and p63
. The PPPPY motif of each sequence is marked in
gray.
View larger version (26K):
[in a new window]
Fig. 4.
Ligand binding specificity governs the
association between YAP and the p73 ,
p73
, and p73
isoforms. A, H1299 cells transiently transfected
with GFP-YAP were extracted and, following a preclearing with
glutathione-agarose beads, were incubated with GST-p73
, GST-p73
,
or GST alone. The blot was probed with an anti-GFP polyclonal serum.
B, cell lysates of H1299 cells transiently transfected with
vectors encoding either p73
(lanes 4-6) or
p73
(lanes 7-9) or with an empty vector
(lanes 1-3) were processed as reported above and
incubated with the indicated GST fusion proteins. The blot was probed
with anti-p73 polyclonal serum. Coomassie staining of replica
gel showing the GST fusion proteins (lower
panel).
View larger version (60K):
[in a new window]
Fig. 5.
Differential binding of YAP to
p63 and p53. A, cell extracts
of H1299 cells transfected with the indicated plasmids were incubated
with GST-p63
(upper panel) or GST alone
(upper middle panels). The blot was
probed with anti-GFP polyclonal serum. The lower
panels show protein levels of GFP-YAP and GFP employed in
the transient transfections. B, identical cell lysates
processed as above reported were incubated with GST-p53 or GST alone.
The blot was probed with anti-GFP polyclonal serum. Coomassie staining
of a replica gel showing the p63
and p53-GST fusion proteins as well
as GST alone are reported in the last panels of
A and B. C, cell extracts derived from
P19 cells were processed as reported in A and incubated with
GST-YAP/WW (lane 3) or GST alone (lane
2). The blot was probed with anti-p53 mAb 421. D,
H1299 cells stably overexpressing inducible human WT p53 were extracted
and, following a preclearing, were incubated with GST-YAP/WW
(lanes 3 and 6) or GST alone
(lanes 2 and 4). The blot was probed
with a mixture of anti-p53 mAbs DO1 and 1801. Coomassie
staining of replica gel showing the GST fusion proteins
(lower panel).
by c-Abl Are
Dispensable for the Association with YAP--
It has recently been
reported that c-Abl interacts physically with p73 (35-37). As a
consequence, p73 is tyrosine-phosphorylated, and its apoptotic activity
is increased (36, 37). In order to investigate whether the binding of
c-Abl to p73 influences the association of p73 with YAP, we employed
pull-down assays.
mutant, which has an alanine
instead of proline at residue 338 (P338A) and is unable to bind to
c-Abl, associated with YAP (36). To this end, H1299 cells were
transiently transfected with a vector encoding p73
-P338A-HA (H-p73
-PA) or with an empty vector (H1299). As shown in Fig. 6A, p73
-P338A-HA was pulled
down only by GST-YAP/WW.
View larger version (33K):
[in a new window]
Fig. 6.
Binding to as well as
tyrosine-phosphorylation of p73 by c-Abl is
dispensable for the association with YAP. H1299 cells were
transiently transfected with the indicated plasmids. Cell extracts (2 mg) were first precleared with glutathione-agarose beads and then
incubated with GST-YAP/WW or GST fusion proteins. The blot was probed
with anti-p73 polyclonal serum. A, binding of p73
P338A
mutant to the above mentioned GST fusion proteins (lanes
2 and 3 and lanes 5 and
6). B, binding of p73
Y99F, p73
Y121F,
p73
Y99F/Y121F mutants to the indicated GST fusion proteins
(lanes 2 and 3, 5 and
6, 8 and 9, and 11 and
12). Aliquots containing 100 µg of total protein from
unprocessed lysates were subjected to immunoblot as previously
reported: lanes 1 and 4 (A); lanes 1, 4,
7, and 10 (B).
mutants carrying substitutions of tyrosine to phenyalanine (Y99F,
Y121F, and Y99F/Y121F) could still associate with YAP (Fig.
6B).
to c-Abl and the
phosphorylation of p73
by c-Abl do not influence the association of
p73 with YAP.
--
It
has previously been reported that YAP itself contains a potent
transactivation domain and stimulates the activity of the transcription
factor, polyomavirus enhancer binding protein 2 (61).
transcriptional activity, we co-transfected H1299 cells with p73
together with GFP-YAP or GFP alone and a luciferase reporter gene driven by the p73-responsive Bax promoter or by the
p73-responsive mdm2 promoter. We report that while a
limiting amount of transfected p73
stimulated the
mdm2 promoter as well as the Bax
promoter activity 1.5-2-fold, respectively, over the control vector,
the stimulation became much more evident when YAP was also added
(GFP-YAP/p73
) (Fig. 7, A
and B).
View larger version (17K):
[in a new window]
Fig. 7.
YAP enhances the transcriptional activity as
well as the induction of Bax protein by
p73 . H1299 cells were transiently
transfected with the indicated combinations of plasmids
encoding p73
(25 ng/60-mm dish) or YAP (100 ng/dish) or pEGFP vector
control together with mdm2 (A) and
Bax (B) luciferase reporter plasmids (50 ng/dish). The total amount of transfected DNA in each dish was keep
constant by the addition of empty vector wherever necessary.
Cell extracts were prepared 36 h later and subjected to
determination of luciferase activity. Results are represented as -fold
induction of luciferase activity compared with the control cells
transfected with an empty pEGFP expression vector. Histograms show the
mean of a typical experiment of three performed in triplicate;
bars indicate S.D. C, H1299 cells were
transiently transfected with the indicated combinations of plasmids
encoding p73
(2 µg/60-mm dish) or YAP (4 µg/60-mm dish) or pEGFP
vector control. Cell extracts (100 µg/lane) were prepared 36 h
later, subjected to SDS-12.5% PAGE, and immunoblotted with anti-Bax
polyclonal serum or with anti-Hsp70 antibody for equal loading.
operated not only on artificial promoter constructs
but also on endogenous p73-responsive chromosomal genes, we analyzed
the levels of Bax protein in cells overexpressing p73
alone or
together with a plasmid encoding human YAP. As shown in Fig.
7C, only in the presence of co-transfected YAP, p73
increases steady state levels of the Bax protein. Thus, YAP can
interact with p73
not only physically but also functionally.
Phe in the PPPPY Motif
of p73
Abolishes the Association with, and Is Not Co-activated by,
YAP--
The structure of a WW domain of human YAP with its cognate
ligand was originally solved by NMR spectroscopy (58). The hallmarks of
the binding pocket of the WW domain of human YAP include three hydrophobic residues, leucine, tyrosine, the second conserved tryptophan, and histidine (58). Two prolines of the ligand
(PPXY) form van der Waals contacts with the second
tryptophan, whereas the terminal tyrosine of the ligand fits into a
hydrophobic pocket. Extensive mutagenesis of the WW domain of YAP and
its cognate ligand and the recent determination of a high resolution
structure of the WW domain of dystrophin in complex with
-dystroglycan also confirmed the requirement of Y in the
PPXY motif for complex formation (44, 59, 60).
in the association with YAP, we generated a GST-p73
mutant in which
the tyrosine in the YAP-WW ligand binding consensus was mutated to
phenyalanine (59). The ability of this mutant to bind to YAP was tested
by pull-down assay. As shown in Fig.
8A (lanes
2-4), endogenous YAP is pulled down by GST-p73
but not by GST-p73
, Y487P, or GST alone.
View larger version (16K):
[in a new window]
Fig. 8.
Point mutation of the terminal tyrosine of
the PPPPY motif of p73 abolishes the binding
with, and is not co-activated by, YAP. A, cell extracts
derived from P19 cells were first precleared with glutathione-agarose
beads and then incubated with the indicated GST fusion proteins
(lanes 2-4). The blot was probed with anti-YAP
polyclonal serum. B, Coomassie staining of a replica gel
showing the p73
and p73
Y487F GST fusion proteins. C,
H1299 cells were transiently transfected with the indicated
combinations of plasmids encoding p73
or p73
Y487P (25 ng/60-mm
dish), YAP (100 ng/dish), or pEGFP vector control together with Bax
luciferase reporter plasmid (50 ng/dish) The total amount of
transfected DNA in each dish was kept constant by the addition of empty
vector wherever necessary. Cell extracts were prepared 36 h later
and subjected to determination of luciferase activity. Results are
represented as -fold induction of luciferase activity compared
with the control cells transfected with an empty pEGFP
expression vector. Histograms show the mean of a typical experiment
performed in triplicate; bars indicate S.D.
. To this end, we assessed the effects of YAP on
the transcriptional activity of the p73
Y487P mutant. As reported in
Fig. 8C, co-expression of YAP, unlike for p73
, does not
promote the transcriptional activity of the p73
mutant measured as
ability to stimulate the p73-responsive Bax promoter.
is crucial for the association between p73
and YAP, and
the terminal tyrosine of that motif is required for the association of
p73
with YAP. Furthermore binding to YAP is required for
co-activation of p73
.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, p73
, and p63
, YAP does not bind to
p53, highlighting the principle of sequence-specific protein-protein
interaction as a determinant of precise biological output(s). A further
level of specificity for the association between p73 and YAP exists
among the different isoforms of p73. Our findings demonstrate that in
contrast to isoforms
and
, p73
does not bind to YAP. Although
a complete comprehension of the functional implications of these
differential interactions requires further analysis, this observation
provides the first step in the understanding of the fine tuning of the
biological output of the p53 family interaction network.
transcriptional
activity. To some extent, YAP is reminiscent of
-catenin, which
takes part in both cell-cell contact mediated by cadherin and in
transcriptional co-activation downstream of Wnt family members as well
as of p53 (67-69). It is reasonable to depict a scenario in which
proline-rich regions of p73 can be recruited by proteins containing SH3
as well as WW domains following the activation of specific signaling
pathways. Our results suggest that the interactions of p73 with Abl and
YAP are independent events, since binding to c-Abl and tyrosine
phosphorylation of p73 are not required for the association with
YAP.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank W.G. Jr Kaelin, G. Melino and X. Espanel for expression plasmids, D. Lane for DO1 antibody and A.M. Salvatori for P19 cells. We are particularly grateful to O. Segatto and O. Monti for helpful suggestions and collaboration.
![]() |
FOOTNOTES |
---|
* This work was supported by Telethon-Italy Grant 369/bi (to G. B.), Associazione Italiana per la Ricerca sul Cancro Telethon and the Consiglio Nazionale delle Ricerche target project in Biotechnology (to G. C.), and by Human Frontier Science Program Organization Grant RG0234 and National Institutes of Health Grants AR45626 and CA45757 (to M. S.).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.
§ These authors contributed equally to this work.
¶ Supported by European Community Grant QLG1-1999-00273.
¶¶ To whom correspondence should be addressed: Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, Via delle Messi d'Oro 156, Rome 00158, Italy. Tel.: 39-06-49852563; Fax: 39-06-49852505; E-mail: blandino@ifo.it.
Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M010484200
2 A. Zucconi, G. Cesareni, manuscript in preparation.
3 B. Cristofawelli and G. Blandino, unpublished observations.
4 G. Fontemaggi and G. Blandino, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: WT, wild type; BES, N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid; YAP, Yes-associated protein; GST, glutathione S-transferase; PCR, polymerase chain reaction; HA, hemagglutinin; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; IB, immunoblot.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Hollstein, M., Soussi, T., Thomas, G., von-Brevern, M., and Bartsch, H. (1997) Recent Res. Cancer Res. 143, 369-389[Medline] [Order article via Infotrieve] |
2. |
Oren, M.
(1999)
J. Biol. Chem.
274,
36031-36034 |
3. | Levine, A. J. (1997) Cell 88, 323-331[Medline] [Order article via Infotrieve] |
4. | Hansen, R., and Oren, M. (1997) Curr. Op. Genet. Dev. 7, 46-51[CrossRef][Medline] [Order article via Infotrieve] |
5. | Almog, N., and Rotter, V. (1997) Biochim. Biophys. Acta 1333, F1-F27[CrossRef][Medline] [Order article via Infotrieve] |
6. | Dulic, V., Kaufmann, W. K., Lees, S. J., Tisty, T. D., Lees, E., Harper, J. W., Elldge, S. J., and Reed, S. (1994) Cell 76, 1013-1023[Medline] [Order article via Infotrieve] |
7. | El-Deiry, W. S., Tokino, S., Velculescu, V. E., Levy, D. B., Parson, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993) Cell 75, 817-825[Medline] [Order article via Infotrieve] |
8. | Miyashita, T., and Reed, J. C. (1995) Cell 80, 293-299[Medline] [Order article via Infotrieve] |
9. | Polyak, K., Xia, Y., Zweier, J. L., Kinzler, K. W., and Vogelstein, B. (1997) Nature 389, 300-305[CrossRef][Medline] [Order article via Infotrieve] |
10. | Kaghad, M., Bonnet, H., Yang, A., Creancier, L., Biscan, J., Valent, A., Minty, A., Chalon, P., Lelias, J., Dumont, X., Ferrara, P., McKeon, F., and Caput, D. (1997) Cell 90, 809-819[Medline] [Order article via Infotrieve] |
11. | Oren, M. (1997) Cell 90, 829-832[Medline] [Order article via Infotrieve] |
12. | Osada, M., Ohba, M., Kawahara, C., Ishioka, C., Kanamaru, R., Katoh, I., Ikawa, Y., Nimura, Y., Nakagawara, A., Obinata, M., and Ikawa, S. (1998) Nat. Med. 4, 839-843[Medline] [Order article via Infotrieve] |
13. | Trink, B., Okami, K., Wu, L., Sriuranpong, V., Jen, J., and Sidransky, D. (1998) Nat. Med. 4, 747[Medline] [Order article via Infotrieve] |
14. | Yang, A., Kaghad, M., Wang, Y., Gillett, E., Fleming, M. D., Dostch, V., Andrews, N. C., Caput, D., and McKeon, F. (1998) Mol. Cell 2, 305-316[Medline] [Order article via Infotrieve] |
15. | Jost, C. A., Marin, C. M., and Kaelin, W. J. (1997) Nature 389, 191-194[CrossRef][Medline] [Order article via Infotrieve] |
16. |
De Laurenzi, V.,
Raschellà, G.,
Barcaroli, D.,
Annichiarico-Petruzzelli, M.,
Ranalli, M.,
Catani, M. V.,
Tanno, B.,
Costanzo, A.,
Levrero, M.,
and Melino, G.
(2000)
J. Biol. Chem.
275,
15226-15231 |
17. |
De Laurenzi, V.,
Costanzo, A.,
Barcaroli, D.,
Terrinoni, A.,
Falco, M.,
Annichiarico-Petruzzelli, M.,
Levrero, M.,
and Melino, G.
(1998)
J. Exp. Med.
188,
1763-1768 |
18. | De Laurenzi, V., Catani, M. V., Costanzo, A., Terrinoni, A., Corazzari, M., Levrero, M., Knight, R. A., and Melino, G. (1999) Cell Death Differ. 6, 389-390[CrossRef][Medline] [Order article via Infotrieve] |
19. | Yang, A., Walker, N., Bronson, R., Kaghad, M., Oosterwegel, M., Bonnin, J., Vagner, C., Bonnet, H., Dikkes, P., Sharpe, A., McKeon, F., and Caput, D. (2000) Nature 404, 99-103[CrossRef][Medline] [Order article via Infotrieve] |
20. | Marin, M. C., and Kaelin, W. G., Jr. (2000) Biochim. Biophys. Acta 1470, 93-100 |
21. |
Marin, M. C.,
Jost, C. A.,
Irwin, M. S.,
DeCaprio, J. A.,
Caput, D.,
and Kaelin, W. G.
(1998)
Mol. Cell. Biol.
18,
6316-6324 |
22. | Dobbelstein, M., and Roth, J. (1998) J. Gen. Virol. 79, 3079-3083[Abstract] |
23. |
Roth, J.,
Konig, C.,
Wienzek, S.,
Weigel, S.,
Ristea, S.,
and Dobbelstein, M.
(1998)
J. Virol.
72,
8510-8516 |
24. | Steegenga, W. T., Shvarts, A., Riteco, N., Bos, J. L., and Jochemsen, A. G. (1999) Mol. Cell. Biol. 9, 3885-3894 |
25. | Bottger, A., Bottger, V., Sparks, A., Liu, W. L., Howard, S. F., and Lane, D. P. (1997) Curr. Biol. 7, 860-869[Medline] [Order article via Infotrieve] |
26. | Haupt, Y., Maya, R., Kazaz, A., and Oren, M. (1997) Nature 387, 296-299[CrossRef][Medline] [Order article via Infotrieve] |
27. | Kubbutat, M. H. G., Jones, S. N., and Voudsen, K. H. (1997) Nature 387, 299-303[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Zeng, X. Y.,
Chen, L. H.,
Jost, C. A.,
Maya, R.,
Keller, D.,
Wang, X. J.,
Kaelin, W. G. J.,
Oren, M.,
Chen, J. D.,
and Lu, H.
(1999)
Mol. Cell. Biol.
19,
3257-3266 |
29. | Balint, E., Bates, S., and Voudsen, K. H. (1999) Oncogene 18, 3923-3929[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Scharnhorst, V.,
Dekker, P.,
von der EB, A. J.,
and Jochemsen, A. G.
(2000)
J. Biol. Chem.
275,
10202-10211 |
31. |
Minty, A.,
Dumont, X.,
Kaghad, M.,
and Caput, D.
(2000)
J. Biol. Chem.
275,
36316-36323 |
32. |
Di Como, C. J.,
Gaiddon, C.,
and Prives, C.
(1999)
Mol. Cell. Biol.
19,
1438-1449 |
33. | Marin, M. C., Jost, C. A., Brooks, L. A., Irwin, M. S., O'Nions, J., Tidy, J. A., James, N., McGregor, J. M., Harwood, C. A., Yulug, I. G., Voudsen, K. H., Allday, M. J., Gusterson, B., Ikawa, S., Hinds, P. W., Crook, T., and Kaelin, W. G. (2000) Nat. Genet. 25, 47-55[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Strano, S.,
Munarriz, E.,
Rossi, M.,
Cristofanelli, B.,
Shaul, Y.,
Castagnoli, L.,
Levine, A. J.,
Sacchi, A.,
Cesareni, G.,
Oren, M.,
and Blandino, G.
(2000)
J. Biol. Chem.
275,
29503-29512 |
35. | Gong, J. G., Costanzo, A., Yang, H. Q., Melino, G., Kaelin, W. G. J., Levrero, M., and Wang, J. Y. J. (1999) Nature 399, 806-809[CrossRef][Medline] [Order article via Infotrieve] |
36. | Agami, R., Blandino, G., Oren, M., and Shaul, Y. (1999) Nature 399, 809-813[CrossRef][Medline] [Order article via Infotrieve] |
37. | Yuan, Z. M., Shioya, H., Ishiko, T., Sun, X. G., Gu, J. J., Huang, Y. Y., Lu, H., Kharbanda, S., Weichselbaum, R., and Kufe, D. (1999) Nature 399, 814-817[CrossRef][Medline] [Order article via Infotrieve] |
38. | Bork, P., and Sudol, M. (1994) Trends Biochem. Sci. 19, 531-533[CrossRef][Medline] [Order article via Infotrieve] |
39. | Sudol, M. (1998) Oncogene 17, 1469-1474[CrossRef][Medline] [Order article via Infotrieve] |
40. | Sudol, M., and Hunter, T. (2000) Cell 103, 1001-1004[Medline] [Order article via Infotrieve] |
41. | Chen, H. I., and Sudol, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7819-7823[Abstract] |
42. | Schild, L., Lu, Y., Gautschi, I., Schneeberger, E., Lifton, R. P., and Rossier, B. C. (1996) EMBO J. 15, 2381-2387[Abstract] |
43. | Rentschler, S., Lim, H., Deiminger, K., Bedford, M. T., Espanel, X., and Sudol, M. (1999) Biol. Chem. 380, 431-442[Medline] [Order article via Infotrieve] |
44. | Huang, X., Poy, F., Zhang, R., Joachimiak, A., Sudol, M., and Eck, M. J. (2000) Nat. Struct. Biol. 8, 634-638[CrossRef] |
45. |
Ermekova, K. S.,
Zambrano, N.,
Linn, H.,
Minopoli, G.,
Gertler, F.,
Russo, T.,
and Sudol, M.
(1997)
J. Biol. Chem.
272,
32869-32877 |
46. |
Bedford, M. T.,
Chan, D. C.,
and Leder, P.
(1997)
EMBO J.
16,
2376-2383 |
47. |
Bedford, M. T.,
Reed, R.,
and Leder, P.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
10602-10607 |
48. |
Komuro, A.,
Saeki, M.,
and Kato, J.
(1999)
J. Biol. Chem.
274,
36513-36519 |
49. |
Waragi, M.,
Lammers, C. H.,
Takeuchi, S.,
Imafuku, I.,
Udagawa, Y.,
Kanazawa, I.,
Kawabata, M.,
Mouradian, M. M.,
and Okazawa, H.
(1999)
Hum. Mol. Genet.
8,
977-987 |
50. | Ranganathan, R., Lu, K. P., Hunter, T., and Noel, J. P. (1997) Cell 89, 875-886[Medline] [Order article via Infotrieve] |
51. |
Lu, P. J.,
Zhou, X. Z.,
Shen, M.,
and Lu, K. P.
(1999)
Science
283,
1325-1328 |
52. | Verdecia, M. A., Bowman, M. E., Lu, K. P., Hunter, T., and Noel, J. P. (2000) Nat. Struct. Biol. 8, 639-643[CrossRef] |
53. | Santi, E., Capone, S., Mennuni, C., Lahm, A., Tramontano, A., Luzzago, A., and Nicosia, A. (2000) J. Mol. Biol. 296, 497-508[CrossRef][Medline] [Order article via Infotrieve] |
54. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , pp. 2.64-2.66, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
55. | Wang, Y., Blandino, G., Oren, M., and Givol, D. (1998) Oncogene 17, 1923-1930[CrossRef][Medline] [Order article via Infotrieve] |
56. | Sudol, M. (1994) Oncogene 9, 2145-2152[Medline] [Order article via Infotrieve] |
57. | Blandino, G., Levine, A. J., and Oren, M. (1999) Oncogene 18, 477-485[CrossRef][Medline] [Order article via Infotrieve] |
58. | Macias, M. J., Hyvonen, M., Baraldi, E., Schultz, J., Sudol, M., Saraste, M., and Oschkinat, H. (1996) Nature 382, 646-649[CrossRef][Medline] [Order article via Infotrieve] |
59. |
Chen, H. I.,
Einbond, A.,
Kwak, S. J.,
Linn, H.,
Koepf, E.,
Paterson, S.,
Kelly, J. W.,
and Sudol, M.
(1997)
J. Biol. Chem.
272,
17070-17077 |
60. | Linn, H., Ermekova, K. S., Rentschler, S., Sparks, A. B., Kay, B. K., and Sudol, M. (1997) Biol. Chem. 378, 531-537[Medline] [Order article via Infotrieve] |
61. |
Yagi, R.,
Chen, L. F.,
Shigesada, K.,
Murakami, Y.,
and Ito, Y.
(1999)
EMBO J.
18,
2551-2562 |
62. |
Pawson, T.,
and Nash, P.
(2000)
Genes Dev.
14,
1027-1047 |
63. | Courtneidge, S. A., Dhand, R., Pilat, D., Twamley, G. M., Waterfield, M. D., and Roussel, M. F. (1993) EMBO J. 12, 943-950[Abstract] |
64. |
Mohler, P. J.,
Kreda, S. M.,
Boucher, R. C.,
Sudol, M.,
Stuttus, M. J.,
and Milgram, S. L.
(1999)
J. Cell Biol.
147,
879-890 |
65. |
Darnell, J. E., Jr.
(1997)
Science
277,
1630-1635 |
66. | Bianchi, E., Denti, S., Granata, A., Bossi, G., Geginat, J., Villa, A., Rogge, L., and Pardi, R. (2000) Nature 404, 617-621[CrossRef][Medline] [Order article via Infotrieve] |
67. | Wodarz, A., and Nusse, R. (1998) Annu. Rev. Cell Dev. Biol. 14, 59-88[CrossRef][Medline] [Order article via Infotrieve] |
68. |
Damalas, A.,
Ben-ze'ev, A.,
Simcha, I.,
Shtutman, M.,
Martinez Leal, J. F.,
Zhurinsky, J.,
Geiger, B.,
and Oren, M.
(1998)
EMBO J.
18,
3054-3063 |
69. |
Peifer, M.,
and Polakis, P.
(2000)
Science
287,
1606-1609 |
70. |
Levrero, M.,
De Laurenzi, V.,
Costanzo, A.,
Sabatini, S.,
Gong, J.,
Wang, J. W. J.,
and Melino, G.
(2000)
J. Cell Sci.
113,
1661-1670 |
71. | Espanel, X., and Sudol, M. (2001) J. Biol. Chem., in press |