From the Laborartory of Molecular Genetics, RIKEN
Tsukuba Institute and the ¶ CREST (Core Research for Evolutional
Science and Technology) Research Project of JST (Japan Science and
Technology Corporation), 3-1-1 Koyadai, Tsukuba,
Ibaraki 305-0074, Japan
Received for publication, August 1, 2000, and in revised form, November 1, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The c-myb proto-oncogene product
(c-Myb) is a sequence-specific DNA-binding protein that functions as a
transcriptional activator. The transcriptional coactivator CREB-binding
protein (CBP) binds via its KIX domain to the activation domain
of c-Myb and mediates c-Myb-dependent transcriptional
activation. CBP possesses intrinsic histone acetyltransferase activity,
and can acetylate not only histones but also certain transcriptional
factors such as GATA1 and p53. Here we demonstrate that the C/H2 domain
of CBP, which is critical for the acetyltransferase activity, also
directly interacts with the negative regulatory domain (NRD) of c-Myb. Consistent with this observation, CBP acetylated c-Myb in
vitro at Lys438 and Lys441 within
the NRD. In addition, CBP acetylated c-Myb in vivo not only
at the sites found in this study but also at the p300-induced acetylation sites reported recently. Replacement of lysine by arginine
at all of these sites dramatically decreased the
trans-activating capacity of c-Myb. The results of
transcriptional activation assays with c-Myb acetylation site mutants
suggested that acetylation of c-Myb at each of these five sites
synergistically enhances c-Myb activity. Mutations of these acetylation
sites reduced the strength of the interaction between c-Myb and CBP.
Thus, acetylation of c-Myb by CBP increases the
trans-activating capacity of c-Myb by enhancing its
association with CBP. These results demonstrate a novel molecular
mechanism of regulation of c-Myb activity.
The c-myb proto-oncogene is the cellular progenitor of
the v-myb oncogenes carried by the chicken retroviruses
avian myeloblastosis virus and E26, which transform
myelomonocytic hematopoietic cells (see Refs. 1 and 2, and for review,
see Ref. 3). Analysis of c-myb-deficient mice and transgenic
mice expressing dominant negative forms of the c-myb gene
product (c-Myb)1 indicated
that c-myb is essential for the proliferation of immature hematopoietic cells and for the development of T cells (4-6). c-Myb is
a transcriptional activator that binds to the specific DNA sequence,
5'-AACNG-3' (7-10). By inducing transcription of a group of target
genes, c-Myb regulates both proliferation and apoptosis of
hematopoietic cells (11-13). c-Myb has three functional domains, which
are aligned from the N terminus in the order: DNA-binding domain,
transcriptional activation domain, and negative regulatory domain (NRD)
(10). The DNA-binding domain in the NH2-terminal region of
c-Myb consists of three imperfect tandem repeats of 51-52 amino acids,
each containing a helix-turn-helix variation motif (14-16). The
transcriptional activation domain of c-Myb, which is rich in acidic
amino acids, is adjacent to the DNA-binding domain. The transcriptional
coactivator CBP (CREB-binding protein) binds to this activation domain
to mediate c-Myb-induced transcriptional activation (17, 18). Deletion
of the NRD, located in the carboxyl-proximal portion of the molecule,
increases both trans-activation and transformation capacity,
implying that this domain normally represses c-Myb activity (10,
19-23), although the mechanism of negative regulation by NRD remains unknown.
CBP was originally identified as a coactivator of the transcription
factor cAMP response element-binding protein (CREB). CBP acts as a
bridging factor between CREB and the general transcription factor TFIIB
(24, 25). The CBP gene family contains at least one other
member, p300, which was originally identified as a protein that binds to the adenovirus E1A protein (26). Both CBP and p300 bind
to the phosphorylated form of CREB and also to E1A (27, 28). CBP binds
not only to phosphorylated CREB but also to nonphosphorylated forms of
many other transcription factors, including c-Myb (17, 18) and nuclear
hormone receptors (29, 30) (for review, see Ref. 31). CBP contributes
to the transcriptional activation mediated by each of these factors.
CBP possesses intrinsic histone acetyltransferase (HAT) activity (32,
33) and binds to other HAT proteins such as PCAF (34) and ACTR (35),
suggesting that it contributes to transcriptional activation by
disrupting repressive chromatin structure via acetylation of histones.
The finding that certain TAFs (TBP (TATA-binding protein)-associated
factors) are integral components of PCAF and yeast SAGA·HAT
complexes further suggests a mechanistic connection between the RNA
polymerase II machinery and CBP HAT activities (36, 37).
CBP/p300 acetylates not only histones but also transcription factors.
p300 directly acetylates p53 and GATA-1, and enhances their
sequence-specific DNA binding activity (38, 39). Similar observations
were reported for E2F (40, 41) and c-Myb (42). In addition to some
sequence-specific DNA-binding proteins, CBP also acetylates other types
of transcription factors. Drosophila CBP acetylates T-cell
factor, a high mobility group domain protein, and this acetylation
blocks the interaction between T-cell factor and its coactivator
Here, we have identified CBP-induced acetylation sites in the c-Myb
protein. Three of the sites correspond to those recently identified as
p300-induced acetylation sites, and the two other sites are new. Our
results suggest that acetylation of c-Myb at each site synergistically
increases the trans-activating capacity of c-Myb.
Acetylation of c-Myb enhanced its affinity for CBP, explaining at least
partly the mechanism of CBP stimulation of the
trans-activating capacity of c-Myb.
Plasmid Construction--
The plasmids pGEX-KIX, pGEX-Bromo,
pGEX-C/H2, and pGEX-C/H3 to express GST fusions containing various
portions of CBP were described previously (45). The expression plasmids
for GST fusion proteins of ATF-2, Smad3/4, and c-Ski were also
described previously (45, 46). To express GST fusion proteins
containing various regions of c-Myb, appropriate DNA fragments of c-Myb
were introduced into the pGEX vectors (Amersham Pharmacia Biotech). The
pSPUTK vector (Stratagene) was used for in vitro
transcription/translation of the various derivatives of c-Myb. The
expression plasmids for the c-Myb mutants were described previously
(10, 22). The polymerase chain reaction-based method (56) was used to
introduce the mutations K438R, K441R, 2K/R, or 5K/R into c-Myb. The CBP expression plasmid pRc/RSV-CBP-HA was a gift from R. Goodman. The
plasmids to express various derivatives of Gal4-c-Myb fusions, in which
the DNA-binding domain of Gal4 was fused to c-Myb, were constructed by
the polymerase chain reaction-based method using the
cytomegalovirus promoter-containing vector pCMX (47).
In Vitro Binding Analysis with GST Fusion Proteins--
The GST
pull-down assay using GST-CBP and in vitro translated c-Myb
was essentially performed as described previously (17). The GST fusion
proteins were expressed in Escherichia coli and bacterial
lysates containing 20 µg of GST-CBP were rocked for 2-3 h at 4 °C
with 100 µl of glutathione-Sepharose beads (Amersham Pharmacia
Biotech). The beads were washed with 1 ml of 0.6 M NaCl two
times, with phosphate-buffered saline containing 0.05% Nonidet P-40
four times, and then with 1 ml of binding buffer (20 mM
Hepes, pH 7.7, 150 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 1% skim milk, 1 mM
dithiothreitol, 0.05% Nonidet P-40). Various forms of
[35S]methionine-labeled c-Myb were synthesized using an
in vitro transcription/translation kit according to the
procedures described by the supplier (Promega). A sample from the
reaction was mixed with GST-CBP affinity resin in 750 µl of binding
buffer. After rocking at 4 °C overnight, the resin was washed with 1 ml of binding buffer 5 times, and resuspended in SDS sample buffer, and
boiled to release bound proteins. The proteins were analyzed by
SDS-PAGE followed by autoradiography.
In Vitro Acetylation Assay using Immunoprecipitated
CBP--
HepG2 cells were transfected with 6 µg of the CBP
expression plasmid pRc/RSV-CBP-HA using LipofectAMINE (Life
Technologies, Inc.). Two days after transfection, cells were disrupted
using lysis buffer (50 mM Hepes, pH 7.5, 250 mM
NaCl, 0.2 mM EDTA, 0.5% Nonidet P-40) containing a
protease inhibitor mixture (Roche Molecular Biochemicals) and
centrifuged at 15,000 rpm for 20 min. The anti-CBP CT polyclonal
antibodies (Upstate Biotech) were added to the supernatant and
incubated on ice overnight. Protein G-Sepharose was added and the
mixture was rotated at 4 °C for 3-4 h. The resulting
immunocomplexes were washed with lysis buffer 3 times and with
acetylation buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40)
containing the protease inhibitors. About 1-2 µg of the purified GST
fusion protein as substrate and 1 µl of [14C]acetyl-CoA
(51 mCi/mmol, Amersham Pharmacia Biotech) were added to the protein
G-Sepharose beads containing CBP, and the reaction volume was adjusted
to 30 µl with acetylation buffer. The samples were incubated at
30 °C for 1 h and analyzed by SDS-PAGE. Gels containing
14C-labeled proteins were fixed with 10% glacial acetic
acid and 40% methanol for 1 h. The efficiency of autoradiography
was enhanced by impregnating the gels with a fluorography enhancing
solution (Amplify, Amersham Pharmacia Biotech) for 30 min. Gels were
then dried and autoradiography was performed.
In Vitro Acetylation Assay using GST-CBP--
The GST-CBP fusion
protein containing the HAT domain (amino acids 1099-1758) was purified
using glutathione-Sepharose beads from E. coli extract. The
acetylation assay was done as described above except that 1 µg of
GST-CBP was used instead of immunoprecipitated CBP as a source of
acetyltransferase enzyme.
In Vivo Acetylation Assay--
To investigate the in
vivo acetylation of c-Myb by CBP, 293 cells were transfected with
3 µg of the CBP expression plasmid pRc/RSV-CBP-HA together with equal
amounts of one of the plasmids expressing various forms of c-Myb. Two
days after transfection, the cells were lysed using RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton
X-100, 1% sodium deoxycholate, 0.1% SDS, 10 mM sodium
butylate) and centrifuged at 15,000 rpm. The supernatant were subjected
to SDS-PAGE and analyzed by Western blotting. The acetylated forms of
c-Myb were detected with anti-acetylated lysine polyclonal antibody
(New England Biolabs), while a mixture of acetylated and nonacetylated
forms of c-Myb were detected by the anti-c-Myb monoclonal antibody 1-1 or 5-1.
Reporter Gene Assay--
The Myb site containing luciferase
reporter, in which the SV40 early promoter is linked to six tandem
repeats of the Myb-binding site, was transfected into HepG2 cells
together with plasmid expressing various forms of c-Myb or the control
vector, and the internal control plasmid pRL-CMV using the
CaPO4 method. Two days after transfection, the cells were
lysed, and luciferase activities were measured by using the dual
luciferase assay system (Promega). Experiments were repeated 4 times,
and the data were averaged. To examine the trans-activation
capacity of the Gal4-Myb fusion, the Gal4 site containing luciferase
reporter, in which the thymidine kinase promoter is linked to three
tandem repeats of the Gal4-binding site, was transfected together with
plasmid expressing various forms of Gal4-Myb fusions or the control
vector and the internal control plasmid pact- Coimmunoprecipitation--
To examine the effect of c-Myb
acetylation on c-Myb affinity for CBP, 293 cells were transfected
together with 3 µg of the CBP expression plasmid pRc/RSV-CBP-HA and 3 µg of plasmid expressing various forms of c-Myb using LipofectAMINE
(Life Technologies). Two days after transfection, cell lysates were
prepared using TNE buffer (10 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 10 mM sodium butyrate) containing a protease inhibitor mixture
and the lysates were centrifuged. The CBP-containing immunocomplexes were precipitated with anti-CBP CT polyclonal antibody from the supernatant and washed with TNE buffer 3 times. The immunocomplexes were separated on 8% SDS gels and analyzed by Western blotting using
the anti-c-Myb monoclonal antibody 5-1 and a chemiluminescent detection
regent (New England Biolabs).
Direct Interaction between the C/H2 Region of CBP and the NRD of
c-Myb--
We previously reported that CBP directly binds to the
transcriptional activation domain of c-Myb via the N-terminal KIX
domain (17). Numerous other transcriptional factors such as CREB also bind to the KIX domain. In a more precise analysis of the interaction between c-Myb and CBP, we obtained evidence suggesting that a region
other than the KIX domain also directly interacts with c-Myb. To
precisely identify this region, we performed the GST pull-down assay
using GST-CBP fusion proteins containing various portions of CBP (Fig.
1). In vitro translated mouse
c-Myb directly bound to not only the GST-KIX fusion but also to the
GST-CBP fusion containing the C/H2 domain. To determine which region of
c-Myb interacts with the C/H2 domain of CBP, we next performed GST
pull-down assays using the GST-C/H2 fusion protein bound to
glutathione-Sepharose beads and various derivatives of c-Myb translated
in vitro (Fig. 2, A
and B). The results indicated that the
NH2-terminal two-thirds of the NRD (amino acids 326-448)
was responsible for binding to GST-C/H2. To further confirm that the
NRD of c-Myb directly binds to the C/H2 domain of CBP, the GST
pull-down assays using the GST fusion containing the NRD were performed
(Fig. 2C). The in vitro translated CBP fragment
containing the C/H2 region (amino acids 1191-1758) bound to the GST
fusion containing the NRD of c-Myb (amino acids 326-448). Thus, in
addition to the previously reported interaction between the KIX domain
of CBP and the transcriptional activation domain of c-Myb, the C/H2
domain of CBP also binds to the NRD of c-Myb.
Acetylation of c-Myb NRD by CBP--
Since the C/H2 domain of CBP
is critical for the intrinsic HAT activity of CBP, the direct
interaction between the C/H2 domain of CBP and c-Myb suggested that CBP
might directly acetylate c-Myb via its HAT domain. To test this,
various transcription factors expressed in bacteria (Fig.
3A) were incubated with the
CBP protein together with [14C]acetyl-CoA, and the level
of acetylation of the transcription factors was investigated (Fig.
3B). The CBP protein was prepared by immunoprecipitating CBP
with the anti-CBP polyclonal antibody from extracts of HepG2 cells
transfected with the mouse CBP expression plasmid. The c-Myb protein
was efficiently acetylated, whereas the other four transcription
factors, ATF-2, Smad3, Smad4, and Ski, were not. To identify which
region of c-Myb molecule was acetylated by CBP, we used a series of
GST-c-Myb fusion proteins containing various portions of c-Myb (Fig.
4B) for in vitro
acetylation assays. Among the five GST-c-Myb fusion proteins, only the
fusion protein containing the NRD was acetylated by CBP (Fig. 4,
A and C). This is consistent with our observation
that the C/H2 domain of CBP directly binds to the NRD of c-Myb.
Identification of the Acetylation Sites in the NRD--
To
determine which lysine residue(s) in the NRD are acetylated by CBP, we
first used a series of GST-NRD fusion proteins containing various
portions of the NRD truncated from the COOH terminus (Fig. 5,A and B). The GST
fusion proteins containing the entire NRD (GST-326-563 and
GST-326-500) were efficiently acetylated by CBP (Fig. 5C, left
panel). Deletion from the COOH terminus to amino acid 449 (GST-326-448) did not abrogate acetylation by CBP, whereas deletion to
amino acid 436 (GST-326-435) dramatically reduced the acetylation
level. Next, we used one GST fusion protein containing the downstream
region from the NRD (GST-501-636) and two GST fusion proteins
containing both the downstream region from the NRD and the different
C-terminal region of the NRD (GST-473-636 and GST-449-636) (Fig.
5C, right panel). Neither of these three GST
fusions were acetylated by CBP. These results indicate that the region
between amino acids 436 and 448 contains the CBP-induced acetylation
site(s).
This narrow region contains two lysine residues, Lys438 and
Lys441, which could serve as potential CBP-mediated
acetylation sites (Fig. 6A).
We mutated either or both of these lysine residues to arginine, and
used them in in vitro acetylation assays containing immunoprecipitated CBP (Fig. 6B). The GST-NRD fusion protein
containing amino acids 326-563 was efficiently acetylated by CBP,
whereas the construct with arginine at either Lys438 or
Lys441 displayed significantly reduced but not total loss
of acetylation by CBP (Fig. 6C). Furthermore, the construct
possessing the two mutated lysine residues was very weakly acetylated
by CBP. To further confirm that CBP really acetylates these two lysine
residues, we examined the acetylation of c-Myb by GST-CBP recombinant
proteins expressed in E. coli (Fig. 6D). The
GST-c-Myb fusion protein containing amino acids 326-563 was
efficiently acetylated by GST-CBP protein containing the HAT domain. In
contrast, GST-CBP acetylated neither the GST-c-Myb mutant protein, in
which both Lys438 and Lys441 were mutated to
ariginine, nor the GST protein alone. Thus, CBP directly acetylates the
c-Myb protein in vitro at Lys438 and
Lys441.
In Vivo Acetylation of Lys438 and Lys441 by
CBP--
We next confirmed that the two acetylation sites identified
in vitro were also acetylated by CBP in vivo.
When the c-Myb expression plasmid was transfected into 293 cells
together with increasing amounts of the CBP expression plasmid, the
acetylated form of c-Myb was detected by the anti-acetylated lysine
antibody (Fig. 7A). The amount
of acetylated c-Myb increased in a dose-dependent manner
with the amount of the CBP expression palsmid. These results indicated
that CBP also acetylates c-Myb in vivo. To investigate which
region of c-Myb is acetylated in vivo, plasmids expressing various derivatives of c-Myb were transfected into 293 cells together with the CBP expression plasmid, and the acetylated form of c-Myb was
detected by the anti-acetylated lysine antibodies (Fig. 7B). The two mutants lacking the DNA-binding domain or the transcriptional activation domain ( Effect of CBP-induced Acetylation on trans-Activating Capacity of
c-Myb--
To examine whether acetylation of c-Myb affects the
trans-activating capacity of c-Myb, we performed
co-transfection assays. The luciferase reporter, in which luciferase
expression was placed under the control of the SV40 early promoter
linked to six tandem repeats of the Myb-biding site, was co-transfected
into HepG2 cells together with the c-Myb expression plasmid. Wild-type
c-Myb stimulated luciferase expression 8.3-fold, whereas the 2K/R and 5K/R mutants stimulated luciferase expression by 6.0- and 4.3-fold, respectively (Fig. 8A). These
results indicate that the CBP-induced acetylation of c-Myb positively
regulates the trans-activating capacity of c-Myb.
Furthermore, acetylation at Lys438 and Lys441
synergistically enhanced the trans-activating capacity of
c-Myb already acetylated at Lys467, Lys476, and
Lys481.
In the study of p300-induced acetylation of c-Myb, it was reported that
p300-mediated acetylation enhanced the DNA binding activity of c-Myb
(42). We also observed that CBP-induced acetylation increased the DNA
binding activity of c-Myb.2 In addition, we observed that
the CBP-mediated acetylation of c-Myb stimulated transcriptional
activation by the Gal4-c-Myb fusion protein (Fig. 8B). The
luciferase reporter containing the Gal4-binding sites was transfected
into HepG2 cells together with the plasmid to express the Gal4 fusion
protein containing full-length c-Myb. The luciferase expression from
this reporter was enhanced 4.5-fold by Gal4-c-Myb. Replacement of both
Lys438 and Lys441 by arginine (2K/R) suppressed
activation by Gal4-Myb to 3.6-fold. Furthermore, replacement of all
five lysine residues by arginines suppressed activation to 2.2-fold.
The expression levels of these acetylation site mutants were similar to
that of wild-type Gal4-c-Myb (Fig. 8C). In the case of the
Gal4-Myb fusion, the Gal4 DNA-binding domain, but not the authentic
DNA-binding domain of c-Myb, was functional. Therefore, these results
indicate that acetylation of c-Myb at five sites specifically
stimulates trans-activation activity.
Acetylation of c-Myb Enhances the Association with CBP--
During
investigation of the mechanism of how CBP-mediated acetylation
increases the trans-activating capacity of c-Myb, we found
that acetylation of c-Myb enhances its association with CBP. As
described above, the two regions of CBP, KIX and C/H2, directly
interact with c-Myb. In the GST pull-down assays using the GST-CBP-KIX
fusion protein, in vitro translated 2K/R and 5K/R mutants
bound to GST-CBP-KIX less efficiently compared with wild-type c-Myb
(Fig. 9A). The 2K/R and 5K/R
mutants bound to GST-CBP-KIX at levels that were 67 and 39% of that of
wild-type c-Myb. In the second GST pull-down assays using GST-CBP-C/H2,
similar results were obtained (Fig. 9B). The binding
efficiencies of the two mutants, 2K/R and 5K/R, to GST-CBP-C/H2 were 61 and 26% of that of the wild-type, respectively. To confirm that c-Myb
acetylation really enhances its affinity for CBP in vivo,
coimmunoprecipitation assays were performed (Fig. 9C). The
plasmid to express the wild-type or the acetylation site mutants was
transfected into 293 cells together with the CBP expression plasmid.
Lysates were prepared from transfected cells, and used for
immunoprecipitation with the anti-CBP antibody. Under these conditions,
apparently less 2K/R and 5K/R mutant proteins were co-precipitated with
CBP compared with wild-type c-Myb. These results indicate that
acetylation of c-Myb increases its affinity for CBP.
In this study, we have identified Lys438 and
Lys441 of c-Myb as the targets of CBP-induced in
vitro acetylation. In addition to theses two lysine residues,
three other lysine residues (Lys467, Lys476,
and Lys481 of mouse c-Myb), which were recently reported to
be acetylated in vitro by p300, were also found to be
acetylated by CBP in vivo. Consistent with our observations,
their study suggested that p300 might also acetylate c-Myb at other
acetylation sites in addition to the three sites at Lys467,
Lys476, and Lys481 on mouse c-Myb. In fact, we
observed that immunoprecipitated p300 acetylated c-Myb in
vitro at Lys438 and Lys441.2
These results strongly suggest that both CBP and p300 acetylate c-Myb
at five lysine residues (Lys438 and Lys441
identified in this study and Lys467, Lys476,
and Lys481 of the recent report). We used the GST-c-Myb
fusion protein as a substrate for in vitro acetylation,
whereas in vitro translated c-Myb was used for acetylation
by p300 (42). This suggests that the three lysine residues identified
as p300-induced acetylation sites may have been masked in the GST-c-Myb
fusion protein by GST-mediated changes in c-Myb protein conformation,
leading to no acetylation at these sites.
Mutation analysis of the acetylation sites in c-Myb suggested that
acetylation at each site synergistically enhanced the
trans-activating capacity of c-Myb. Our results indicated
that acetylation of c-Myb by CBP enhanced its affinity for CBP.
Acetylation of the NRD in c-Myb enhanced its binding not only for the
C/H2 domain of CBP but also for the KIX domain of CBP. The C/H2 domain
interacts with the NRD containing the acetylated lysines, whereas the
KIX domain directly interacts with the activation domain of c-Myb which
lacks acetylation sites. Compared with wild-type c-Myb, the c-Myb
mutant lacking the activation domain ( Both A-Myb and B-Myb, other members of the myb gene family,
are also acetylated by CBP.2 Five lysine residues of mouse
c-Myb (Lys438, Lys441, Lys467,
Lys476, and Lys481), which can be acetylated by
CBP, are well conserved in A-Myb. Both c-Myb and A-Myb bind to CBP to
elicit strong transcriptional activation. Among these five lysine
residues, Lys438 is the only one not conserved in B-Myb,
although the other four lysines are present. This difference could be
partly related to the observation that B-Myb is a weaker
transcriptional activator compared with c-Myb and A-Myb (48, 49).
Although B-Myb has a transcriptional activation domain, which is rich
in acidic amino acids, the COOH-terminal portion containing the
putative acetylation sites appears to be required for transcriptional
activation by B-Myb (50, 51). Cyclin A/Cdk2 phosphoryates this region,
and stimulates its trans-activating capacity (52-55).
Further study will be required to investigate the interesting
possibility that acetylation and phosphorylation of this region
synergistically modulate B-Myb activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin/Armadillo (43). Acetylation of another high mobility group
protein, HMG-I, also leads to transcriptional repression of the
interferon-
gene promoter (44). Thus, CBP appears to control
transcription by acetylating certain transcription factors through
multiple mechanisms.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal. The transfection
efficiency was normalized with
-galactosidase activity. The
luciferase activities were measured as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Fig. 1.
Direct binding of the C/H2 domain of CBP to
c-Myb. A, schematic representation of the GST-CBP
fusion proteins used. On the top, the domain structure of
murine CBP is shown schematically: C/H, cysteine- and
histidine-rich domain; KIX, CREB-binding domain;
Bromo, bromo domain. Structures of the four GST-CBP fusion
proteins used are shown below. The results of the binding
assays shown in C are indicated on the right. B,
analysis of the GST-CBP fusion proteins. The GST-CBP fusion proteins
bound by the glutathione-Sepharose resin were analyzed by 10% SDS-PAGE
followed by Coomassie Brilliant Blue staining. C, binding of
c-Myb to various types of GST-CBP fusion proteins. The
35S-labeled wild-type c-Myb was synthesized in
vitro and mixed with various GST-CBP fusion proteins, and bound
proteins were analyzed by SDS-PAGE followed by autoradiography. The
amount of c-Myb in the input lane was 10% of that used for the binding
assay.
View larger version (23K):
[in a new window]
Fig. 2.
Direct interaction between the C/H2 domain of
CBP and the NRD of c-Myb. A, the functional
domains of c-Myb and the deletion mutants used are schematically shown.
The results of binding assays shown in B are indicated on
the right. B, GST pull-down assay. In the input lanes,
various forms of 35S-c-Myb were synthesized in
vitro and analyzed by SDS-PAGE followed by autoradiography. In the
right panel, the 35S-c-Myb proteins indicated
above each lane were mixed with the GST-CBP-C/H2 affinity
resin and the bound proteins were analyzed. In the input lane, the
amount of protein was 10% of that used in the binding assay.
C, GST pull-down assay using GST-NRD. Binding of the
in vitro translated CBP fragment containing the HAT domain
(amino acids 1191-1758) was analyzed using the GST-Myb fusion proteins
containing various regions of c-Myb indicated above each lane. In the
input lane, the amount of protein was 10% of that used in the binding
assay.
View larger version (21K):
[in a new window]
Fig. 3.
In vitro acetylation of c-Myb by
CBP. A, analysis of GST fusion proteins. The various
GST fusion proteins bound to glutathione-Sepharose resin or bacterial
lysates containing full-length c-Myb were analyzed by 10% SDS-PAGE
followed by Coomassie Brilliant Blue staining. B, in vitro
acetylation of c-Myb. The CBP expression plasmid was transfected into
HepG2 cells, and cell extracts were prepared and immunoprecipitated
with anti-CBP polyclonal antibodies. The immunoprecipitated CBP was
incubated with various proteins shown above each lane and
[14C]acetyl-CoA. The reaction products were separated by
SDS-PAGE and visualized by autoradiography. Below, the
autoacetylation level of CBP indicates that similar amounts of CBP of
was deposited in each lane.
View larger version (35K):
[in a new window]
Fig. 4.
Localization of the acetylation site in
NRD. A, schematic representation of the GST-c-Myb
proteins used. The results of the acetylation assays shown in
C are indicated on the right. B, analysis of
GST-Myb fusion proteins. Various regions of c-Myb were expressed as GST
fusions, and the proteins bound to glutathione-Sepharose resin or
bacterial lysates containing full-length c-Myb (FL) were
analyzed by 10% SDS-PAGE followed by Coomassie Brilliant Blue
staining. C, in vitro acetylation of GST-c-Myb. The
GST-c-Myb fusion proteins were used for in vitro acetylation
with CBP as described in the legend to Fig. 3B.
View larger version (28K):
[in a new window]
Fig. 5.
Localization of the acetylation sites between
amino acids 436 and 448. A, schematic representation of
the GST-c-Myb proteins used. The results of the in vitro
acetylation assays shown in C are indicated on the
right. B, analysis of GST fusion proteins. The GST-c-Myb
fusion proteins eluted from the glutathione-Sepharose resin were
separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue.
C, in vitro acetylation of GST-c-Myb by CBP. The GST-c-Myb
fusion proteins were used for in vitro acetylation with CBP
as described in the legend to Fig. 3B.
View larger version (33K):
[in a new window]
Fig. 6.
Identification of lysine residues acetylated
by CBP. A, two lysine residues in the region between
amino acids 436 and 448 of c-Myb. The sequence of the 436-448 amino
acid region is shown. Three mutants, in which either or both of the two
lysines were mutated to arginines, were generated. B,
analysis of GST fusion proteins. The GST-c-Myb fusion proteins shown
above each lane were bound to glutathione-Sepharose resin,
separated by 10% SDS-PAGE, and stained with Coomassie Brilliant Blue.
C, in vitro acetylation of GST-Myb by CBP. The
in vitro acetylation of GST-c-Myb fusion proteins shown
above each lane was performed as described in the legend to
Fig. 3B. D, in vitro acetylation of GST-c-Myb by GST-CBP.
The proteins indicated above each lane were incubated with
GST-CBP containing the HAT domain of CBP (amino acids 1099-1758) and
[14C]acetyl-CoA, and acetylated GST-Myb was detected by
autoradiography
DBD or
TA) were not acetylated by CBP.
The DNA-binding domain contains the nuclear localization signals and the
DBD mutant cannot enter the nucleus (10), which means that the
DBD mutant would not be able to associate with CBP. The
transcriptional activation domain is critical for association with CBP
and the
TA mutant has probably much lower affinity for CBP compared
with wild-type c-Myb (17). Thus, failure to detect acetylated forms of
these two mutants may not be due to defects in their acetylation sites,
but rather due to inefficient association between c-Myb and CBP. In
fact, we observed CBP acetylated in vivo the c-Myb
DBD
mutant fused to the Gal4 DNA-binding domain which has a nuclear localization signal.2 CT1 was
efficiently acetylated in vivo, whereas CT2 was not. These
results indicate that the region between amino acids 404 and 500 in the
NRD contains the acetylation sites. These results are consistent with
the data from the in vitro acetylation assays described
above. When the c-Myb mutant in which Lys438 or
Lys441 was replaced by arginine (K438R or K441R) was
expressed together with CBP, the density of the band corresponding to
the acetylated form of c-Myb was significantly lower than that of
wild-type c-Myb (Fig. 7C, left panel). However,
the acetylated form of these two c-Myb mutants could still be detected.
Furthermore, when the c-Myb mutant in which both Lys438 and
Lys441 were replaced by arginine (2K/R) was used, the
density of the band corresponding to the acetylated form of c-Myb was
about half of that of wild-type c-Myb (Fig. 7C, right
panel). Although the results indicate that Lys438 and
Lys441 are acetylated by CBP in vivo, other
sites of acetylation must exist to explain the residual level of
acetylation of the 2K/R mutant. During the course of this study,
another group reported the acetylation of three other sites,
Lys471, Lys480, and Lys485, in the
human c-Myb protein by p300 (42). These three sites in human c-Myb
correspond to Lys467, Lys476, and
Lys481 in mouse c-Myb, and are located further downstream
from the two acetylation sites identified here. Based on this report,
we speculated that the acetylation of the 2K/R mutant occurred at these
three sites. To confirm this possibility, we constructed a mutant c-Myb that had all five lysine residues (two sites identified in this study
and the three sites reported by the other group) mutated to arginines
(5K/R), and used it in the in vivo acetylation assay (Fig.
7C, right panel). The mutant displayed almost no
acetylation by CBP, suggesting that CBP acetylates c-Myb in
vivo at five sites. The use of different substrates for in
vitro acetylation by our group and the other group may explain the
discrepancy in the number of detected acetylation sites (see
"Discussion").
View larger version (30K):
[in a new window]
Fig. 7.
In vivo acetylation of
Lys438 and Lys441 of c-Myb by CBP.
A, c-Myb is acetylated by CBP in vivo. The c-Myb
expression plasmid was transfected into 293 cells together with
increasing amounts of the CBP expression plasmid. Cell lysates were
prepared and analyzed by SDS-PAGE, followed by Western blotting using
the anti-acetylated lysine antibody (top panel), the
anti-c-Myb antibody (middle panel), or the anti-CBP antibody
(bottom panel), respectively. Asterisk shows the
endogenous proteins in 293 cells which were recognized by the
anti-acetylated lysine antibody. B, localization of in
vivo acetylation site in the NRD of c-Myb. The plasmid to express
the indicated c-Myb derivatives was transfected into 293 cells together
with the CBP expression plasmid. Cell lysates were prepared and Western
blotting was performed using the anti-c-Myb antibody (lower left
panel) or anti-acetylated lysine antibody (lower right
panel), respectively. C, five lysine residues including
both Lys438 and Lys441 are acetylated by CBP
in vivo. The plasmid to express wild-type c-Myb
or the acetylation site mutants indicated above each lane
was transfected into 293 cells together with the CBP expression vector
or control vector. Cell lysates were prepared and analyzed by SDS-PAGE,
followed by Western blotting using anti-acetylated lysine antibody
(upper panel) or anti-c-Myb antibody (lower
panel).
View larger version (21K):
[in a new window]
Fig. 8.
Transcripional activation by acetylation site
mutants of c-Myb. A, effect of acetylation site
mutations on transcriptional activation by c-Myb. HepG2 cells were
transfected with a mixture of the Myb site containing luciferase
reporter plasmid and the plasmid to express the indicated derivatives
of c-Myb or the control empty vector, and the luciferase assays were
performed. Experiments were repeated four times and the average level
of transcriptional activating capacity of each form of c-Myb is
indicated in the bar graph along with standard deviations.
B, effect of acetylation site mutations on the
transcriptional activation by Gal4-c-Myb fusions. The Gal4 site
containing luciferase reporter was transfected into HepG2 cells
together with the plasmid to express the Gal4-c-Myb fusions containing
the indicated derivatives of c-Myb or the control vector encoding
Gal4-CT5 shown in C, and the luciferase assays were
performed. C, Western blotting of the Gal4-c-Myb fusion
proteins. HepG2 cells were transfected as described in B,
and the lysates were prepared from an aliquot of cells and analyzed by
SDS-PAGE followed by Western blotting with anti-Gal4 monoclonal
antibody (RK5C1, Santa Cruz). The structures of the Gal4-Myb fusions
are schematically shown above.
View larger version (29K):
[in a new window]
Fig. 9.
Decreased affinity between the acetylation
site mutants of c-Myb and CBP. A, effect of the
acetylation site mutation on the affinity of c-Myb for the KIX domain
of CBP. The 35S-labeled wild-type and mutant c-Myb proteins
were synthesized in vitro and mixed with the GST-CBP-KIX
affinity resin, and bound proteins were analyzed by SDS-PAGE followed
by autoradiography. In the input lanes, the amount of protein was 10%
of that used for the binding assay. Experiments were repeated three
times and the average amount of c-Myb bound to GST-CBP-KIX is indicated
in the bar graph along with standard deviations on the
right. B, effect of the acetylation site mutations in c-Myb
on the affinity of c-Myb for the C/H2 domain of CBP. The
35S-labeled wild-type and mutant c-Myb proteins were
synthesized in vitro and mixed with the GST-CBP-C/H2
affinity resin, and bound proteins were analyzed by SDS-PAGE followed
by autoradiography. Experiments were repeated three times and the
average amount of c-Myb bound to GST-CBP-C/H2 is indicated in the
bar graph along with standard deviations on the right.
C, effect of the acetylation site mutations in c-Myb on the
in vivo affinity of c-Myb for CBP. The plasmid to express
wild-type and mutant c-Myb was transfected into 293 cells together with
the CBP expression plasmid. Lysates were prepared from transfected
cells, and used for immunoprecipitation with anti-CBP antibody. The
immunocomplex was analyzed by SDS-PAGE followed by Western blotting
with anti-Myb or anti-CBP antibody. In the right panels, an
aliquot of lysate was directly used for Western blotting with anti-Myb
or anti-CBP antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TA) has a lower affinity for
the C/H2 domain of CBP (Fig. 2B). These results suggest that
NRD and the activation domain interact with each other in the c-Myb
molecule and synergistically stimulate c-Myb association with CBP.
Probably, acetylation at NRD affects the protein conformation of the
activation domain of c-Myb, leading to enhanced association of c-Myb
with the KIX domain. After binding of c-Myb to the enhancer region of
c-Myb target genes, CBP may be recruited to c-Myb on the enhancer.
However, this association between c-Myb and CBP may be transient or
very weak. Once c-Myb is acetylated by CBP, CBP may become tightly
associated with c-Myb, and mediate more efficient c-Myb-induced
transcriptional activation. In the study of p300-induced acetylation of
c-Myb, it was shown that acetylation of c-Myb enhanced the DNA affinity
of c-Myb for the Myb recognition sequence (42). We also observed that
the DNA-binding capacity of c-Myb was enhanced by CBP.2
Therefore, acetylation of c-Myb by CBP/p300 has two functional consequences for c-Myb activity: increased DNA binding activity and
increased activation potential. Recently, it was also reported that
acetylation of E2F stimulates E2F activity via multiple mechanisms including increased DNA binding activity, activation potential, and
protein half-life (41). Thus, acetylation may affect multiple characteristics of transcription factors. NRD negatively regulates c-Myb activity by associating with uncharacterized inhibitor(s). Thus,
acetylation of the NRD of c-Myb could also serve to inhibit the binding
of such inhibitors to c-Myb.
![]() |
ACKNOWLEDGEMENT |
---|
We thank R. H. Goodman for the gift of the mouse CBP expression plasmid.
![]() |
FOOTNOTES |
---|
* 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.
§ Supported by the Special Researcher's Basic Science Program.
To whom correspondence should be addressed: Laboratory of
Molecular Genetics, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan. Tel.: 81-298-36-9031; Fax: 81-298-36-9030; E-mail: sishii@rtc.riken.go.jp.
Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M006896200
2 Y. Sano, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: c-Myb, myb gene product; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; NRD, negative regulatory domain; HAT, histone acetyltransferase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; RSV, Rous sarcoma virus.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Roussel, M., Saule, S., Lagrou, C., Beug, H., Graf, T., and Stehelin, D. (1979) Nature 281, 452-455[Medline] [Order article via Infotrieve] |
2. | Klempnauer, K.-H., Gonda, T. J., and Bishop, J. M. (1982) Cell 31, 453-463[Medline] [Order article via Infotrieve] |
3. | Lipsick, J. S., and Wang, D. M. (1999) Oncogene 18, 3047-3055[CrossRef][Medline] [Order article via Infotrieve] |
4. | Mucenski, M. L., McLain, K., Kier, A. B., Swerdlow, S. H., Schereiner, C. M., Miller, T. A., Pietryga, D. W., Scott, W. J., and Potter, S. S. (1991) Cell 65, 677-689[Medline] [Order article via Infotrieve] |
5. | Badiani, P., Corbella, P., Kioussis, D., Marvel, J., and Weston, K. (1994) Genes Dev. 8, 770-782[Abstract] |
6. |
Allen, R. D., III,
Bender, T. P.,
and Siu, G.
(1999)
Genes Dev.
13,
1073-1078 |
7. | Biedenkapp, H., Borgmeyer, U., Sippel, A. E., and Klempnauer, K.-H. (1988) Nature 335, 835-837[CrossRef][Medline] [Order article via Infotrieve] |
8. | Ness, S. A., Marknell, A., and Graf, T. (1989) Cell 59, 1115-1125[Medline] [Order article via Infotrieve] |
9. | Weston, K., and Bishop, J. M. (1989) Cell 58, 85-93[Medline] [Order article via Infotrieve] |
10. | Sakura, H., Kanei-Ishii, C., Nagase, T., Nakagoshi, H., Gonda, T. J., and Ishii, S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5758-5762[Abstract] |
11. | Kowenz-Leutz, E., Herr, P., Niss, K., and Leutz, A. (1997) Cell 91, 185-195[Medline] [Order article via Infotrieve] |
12. | Frampton, J., Ramqvist, T., and Graf, T. (1996) Genes Dev. 10, 2720-2731[Abstract] |
13. | Taylor, D., Badiani, P., and Weston, K. A. (1996) Genes Dev. 10, 2732-2744[Abstract] |
14. | Tanikawa, J., Yasukawa, T., Enari, M., Ogata, K., Nishimura, Y., Ishii, S., and Sarai, A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9320-9324[Abstract] |
15. | Ogata, K., Morikawa, S., Nakamura, H., Sekikawa, A., Inoue, T., Kanai, H., Sarai, A., Ishii, S., and Nishimura, Y. (1994) Cell 79, 639-648[Medline] [Order article via Infotrieve] |
16. | Ogata, K., Morikawa, S., Nakamura, H., Hojo, H., Yoshimura, S., Zhang, R., Aimoto, S., Ametani, Y., Hirata, Z., Sarai, A., Ishii, S., and Nishimura, Y. (1995) Nat. Struct. Biol. 2, 309-320[Medline] [Order article via Infotrieve] |
17. | Dai, P., Akimaru, H., Tanaka, Y., Hou, D.-X., Yasukawa, T., Kanei-Ishii, C., Takahashi, T., and Ishii, S. (1996) Genes Dev. 10, 528-540[Abstract] |
18. | Oelgeschlager, M., Janknecht, R., Krieg, J., Schreek, S., and Luscher, B. (1996) EMBO J. 15, 2771-2780[Abstract] |
19. | Gonda, T. J., Buckmaster, C., and Ramsay, R. G. (1989) EMBO J. 8, 1777-1783[Abstract] |
20. | Hu, L.-Y., Ramsay, R. G., Kanei-Ishii, C., Ishii, S., and Gonda, T. J. (1991) Oncogene 6, 1549-1553[Medline] [Order article via Infotrieve] |
21. | Grasser, F. A., Graf, T., and Lipsick, J. S. (1991) Mol. Cell. Biol. 11, 3987-3996[Medline] [Order article via Infotrieve] |
22. | Kanei-Ishii, C., Macmillan, E. M., Nomura, T., Sarai, A., Ramsay, R. G., Aimoto, S., Ishii, S., and Gonda, T. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3088-3092[Abstract] |
23. | Dubendorff, J. W., Whittaker, L. J., Eltman, J. T., and Lipsick, J. S. (1992) Genes Dev. 6, 2524-2535[Abstract] |
24. | Chrivia, J. C., Kwok, R. P. S., Lamb, N., Hagiwara, M., Montminy, M. R., and Goodman, R. H. (1993) Nature 365, 855-859[CrossRef][Medline] [Order article via Infotrieve] |
25. | Kwok, R. P. S., Lundblad, J. R., Chrivia, J. C., Richards, J. P., Bachinger, H. P., Brennan, R. G., Roberts, S. G. R., Green, M. R., and Goodman, R. H. (1994) Nature 370, 223-226[CrossRef][Medline] [Order article via Infotrieve] |
26. | Eckner, R., Ewen, M. E., Newsome, D., Gerdes, M., DeCapiro, J. A., Lawrence, J. B., and Livingston, D. M. (1994) Genes Dev. 8, 869-884[Abstract] |
27. | Lundblad, J. R., Kwok, R. P. S., Laurance, M. E., Harter, M. L., and Goodman, R. H. (1995) Nature 374, 85-88[CrossRef][Medline] [Order article via Infotrieve] |
28. | Arany, Z., Newsome, D., Oldread, E., Livingston, D. M., and Eckner, R. (1995) Nature 374, 81-84[CrossRef][Medline] [Order article via Infotrieve] |
29. | Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., Lin, S.-C., Heyman, R. A., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1996) Cell 85, 403-414[Medline] [Order article via Infotrieve] |
30. | Chakravarti, D., LaMorte, V. J., Nelson, M. C., Nakajima, T., Schulman, I. G., Juguilon, H., Montminy, M., and Evans, R. M. (1996) Nature 383, 99-103[CrossRef][Medline] [Order article via Infotrieve] |
31. | Giles, R. H., Peters, D. J., and Breuning, M. H. (1998) Trends Genet. 14, 178-183[CrossRef][Medline] [Order article via Infotrieve] |
32. | Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H., and Nakatani, Y. (1996) Cell 87, 953-959[Medline] [Order article via Infotrieve] |
33. | Bannister, A. J., and Kouzarides, T. (1996) Nature 384, 641-643[CrossRef][Medline] [Order article via Infotrieve] |
34. | Yang, X.-J., Ogryzko, V. V., Nishikawa, J., Howard, B. H., and Nakatani, Y. (1996) Nature 382, 319-324[CrossRef][Medline] [Order article via Infotrieve] |
35. | Chen, H., Lin, R. J., Schiltz, R. L., Chakravarti, D., Nash, A., Nagy, L., Privalsky, M. L., Nakatani, Y., and Evans, R. M. (1997) Cell 90, 569-580[Medline] [Order article via Infotrieve] |
36. | Ogryzko, V. V., Kotani, T., Zhang, X., Schlitz, R. L., Howard, T., Yang, X. J., Howard, B. H., Qin, J., and Nakatani, Y. (1998) Cell 94, 35-44[Medline] [Order article via Infotrieve] |
37. | Grant, P. A., Schieltz, D., Pray-Grant, M. G., Steger, D. J., Reese, J. C., Yates, J. R., III, and Workman, J. L. (1998) Cell 94, 45-53[Medline] [Order article via Infotrieve] |
38. | Gu, W., and Roeder, R. G. (1997) Cell 90, 595-606[Medline] [Order article via Infotrieve] |
39. | Boyes, J., Byfield, P., Nakatani, Y., and Ogryzko, V. (1998) Nature 396, 594-598[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Marzio, G.,
Wagener, C.,
Gutierrez, M. I.,
Cartwright, P.,
Helin, K.,
and Giacca, M.
(2000)
J. Biol. Chem.
275,
10887-10892 |
41. |
Martinez-Balbas, M. A.,
Bauer, U. M.,
Nielsen, S. J.,
Brehm, A.,
and Kouzarides, T.
(2000)
EMBO J.
19,
662-671 |
42. | Tomita, A., Towatari, M., Tsuzuki, S., Hayakawa, F., Kosugi, H., Tamai, K., Miyazaki, T., Kinoshita, T., and Saito, H. (2000) Oncogene 19, 444-451[CrossRef][Medline] [Order article via Infotrieve] |
43. | Waltzer, L., and Bienz, M. (1998) Nature 395, 521-525[CrossRef][Medline] [Order article via Infotrieve] |
44. | Munshi, N., Merika, M., Yie, J., Senger, K., Chen, G., and Thanos, D. (1998) Mol. Cell 2, 457-467[Medline] [Order article via Infotrieve] |
45. |
Sano, Y.,
Tokitou, F.,
Dai, P.,
Maekawa, T.,
Yamamoto, T.,
and Ishii, S.
(1998)
J. Biol. Chem.
273,
29098-29105 |
46. |
Sano, Y.,
Harada, J.,
Tashiro, S.,
Gotoh-Mandeville, R.,
Maekawa, T.,
and Ishii, S.
(1999)
J. Biol. Chem.
274,
8949-8957 |
47. | Umesono, K., Murakami, K. K., Thompson, C. C., and Evans, R. M. (1991) Cell 65, 1255-1266[Medline] [Order article via Infotrieve] |
48. |
Mizuguchi, G.,
Nakagoshi, H.,
Nagase, T.,
Nomura, N.,
Date, T.,
Ueno, Y.,
and Ishii, S.
(1990)
J. Biol. Chem.
265,
9280-9284 |
49. |
Nakagoshi, H.,
Takemoto, Y.,
and Ishii, S.
(1993)
J. Biol. Chem.
268,
14161-14167 |
50. | Tashiro, S., Takemoto, Y., Handa, H., and Ishii, S. (1995) Oncogene 10, 1699-1707[Medline] [Order article via Infotrieve] |
51. |
Oh, I. H.,
and Reddy, E. P.
(1998)
Mol. Cell. Biol.
18,
499-511 |
52. |
Sala, A.,
Kundu, M.,
Casella, I.,
Engelhard, A.,
Calabretta, B.,
Grasso, L.,
Paggi, M. G.,
Giordano, A.,
Watson, R. J.,
Khalili, K.,
and Peschle, C.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
532-536 |
53. | Lane, S., Farlie, P., and Watson, R. (1997) Oncogene 14, 2445-2453[CrossRef][Medline] [Order article via Infotrieve] |
54. | Ziebold, U., Bartsch, O., Marais, R., Ferrari, S., and Klempnauer, K.-H. (1997) Curr. Biol. 7, 253-260[Medline] [Order article via Infotrieve] |
55. | Saville, M. K., and Watson, R. J. (1998) Oncogene 17, 2679-2689[CrossRef][Medline] [Order article via Infotrieve] |
56. | Higuchi, R. (1990) in PCR Technology: Principles and Applications for DNA Amplification (Erlich, H. A., ed) , pp. 61-70, Stockton Press, New York |