From the Department of Biochemistry and Molecular Biology and the
Vollum Institute, Oregon Health Science
University, Portland, Oregon 97201
Received for publication, October 12, 2000, and in revised form, October 31, 2000
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ABSTRACT |
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We previously reported that p73, like p53,
utilizes p300 or cAMP-response element-binding protein-binding
protein as its coactivator. Here, we extended this work by
further examining whether the intrinsic acetylase activity of p300 is
necessary for stimulating p73 function. Although p300 acetylated the
C-terminal fragment of p73 (amino acids 311-636) in vitro,
it was unable to efficiently acetylate the full-length p73.
Consistently, p300 did not acetylate p73 in vivo when both
the proteins were overexpressed in cells. Also, an acetylase-defective
mutant p300 named p300AT2 was able to elevate p73-dependent
transcription in cells. p300 associated with p73 when forming
DNA-protein complexes and stabilized p73 proteins. These results
demonstrate that p300 does not need its acetylase activity to be a
coactivator of p73.
p300 and CBP1 have been
shown to mediate transcription by many different transcriptional
activators (1), including the tumor suppressor p53 protein (2-4), and
to be involved in cell growth control and neoplasia (see Ref. 5 and
references therein). p300 and CBP are two individual proteins encoded
by two different genes (6-8), but they share a significant homology in
their functional domains with similar biochemical functions. However,
their roles in development and neoplasia are not redundant (5, 9),
indicating that each of them is crucial for cell growth control.
Interestingly, both of these coactivators possess intrinsic acetylase
activity, which plays a critical role in regulating the functions of
some p300/CBP interacting transcriptional activators (10), such as p53
(11, 12). By acetylating p53, p300/CBP stimulates its ability to bind
to DNA in a sequence-specific fashion in vitro (11) and
enhances its transcription in vivo (2-4), indicating that
this group of coactivators participate in the p53 responsive pathway.
Indeed, DNA damage signals stimulate p53 acetylation by p300 (12),
which can be inhibited by the cellular p53 repressor MDM2 (13).
Hence, participating in the p53 pathway represents one role of p300/CBP
in preventing neoplasia.
p300 has also been shown to regulate the function of the human p53
homolog p73 (14, 15), which has several alternate splice forms (16).
p73 shares the sequence homology and some biochemical activity with p53
(14-17). Although p73 was shown to induce the same subset of genes as
p53 does with a minor difference (14, 17, 18), they appear to play
different physiological roles (19). p73-deficient mice displayed
neurological, pheromonal, and inflammatory defects without spontaneous
tumors (19), whereas p53 knockout mice developed normally with an early
onset of tumorigenesis (20). However, because of the high mortality of
the p73 null mice (19), it is difficult to assess the role of p73 in
tumorigenesis. Other studies showed that the oncogenic p53 mutants
inhibit p73 function (21, 22), and the alternate splice form of p73
that lacks the N-terminal transactivation domain represses the function of both p53 and p73 (23, 24), suggesting that p73 may also be involved
in tumorigenesis. However, this function of p73 may be suppressed by
its N-terminal truncated alternates (23, 24), which were found to be
expressed at the high level in some primary tumors (25-28).
Distinct roles of p73 and p53 in development and tumorigenesis suggest
that these proteins may be differentially regulated through separate
mechanisms. Indeed, upon In an attempt to elucidate the mechanisms of p73 regulation, we
previously reported that p73, like p53, utilizes p300 or CBP as its
coactivator. Here, we extended this work by further examining whether
the intrinsic acetylase activity of p300 is necessary for stimulating
p73 function. Surprisingly, p300 was unable to acetylate the
full-length p73 Cell Culture--
Human lung small cell carcinoma H1299 cells
were cultured as described previously (45).
Antibodies and Reagents--
The anti-acetylated lysine antibody
and the polyclonal anti-p63 antibody were purchased from Santa Cruz
Biotechnology Inc. Monoclonal anti-p53 antibody Pab421 was described
previously (14). Polyclonal anti-p73 antibodies were raised
specifically against the C terminus (aa 401-636) of p73 Purification of Recombinant p300, p300AT2, p53, p73 Acetylation Assay--
Acetylation assays were carried out
according to the published method (11, 13). 20 µl of reaction mixture
contained 50 mM Tris-Hcl (pH 8.0), 10% glycerol (v/v), 0.1 mM EDTA, 1 mM dithiothreitol, 10 mM
sodium butyrate, [1-14C]acetyl-CoA, or 500 nM
or different concentrations of acetyl-CoA (Sigma), different amounts of
p53, p73, the p73 C-terminal fragment (aa 311-636), histones, p300, or
p300AT2 (see figure legends for the amounts of the proteins used in
each reaction). The mixture was incubated at 30 °C for 60 min and
analyzed by SDS polyacrylamide gel electrophoresis. Acetylated p53 was
detected either by autoradiography or Western blot analysis using the
polyclonal anti-acetylated lysine antibody (Upstate Biotechnology,
Inc.). Monoclonal anti-p53 antibody 421, polyclonal anti-p73
antibody, and polyclonal anti-p300 antibody were used to detect
corresponding protein levels.
EMSA--
This assay was conducted as described. Proteins, as
indicated in the figure legends, were pre-incubated with antibodies
against p300 or p73 or p63 in the presence or absence of acetyl-CoA, at 30 °C for 30 min, as described above, prior to being mixed with a
DNA binding mixture containing 10 mM Hepes buffer (pH 7.5), 4 mM MgCl2, 60 mM NaCl, 0.1 µg
poly(dI-dC), 0.1% Nonidet P-40, 0.1 mM EDTA, and 5'/3'
32P end-labeled DNA fragments harboring two copies of the
p53RE sequence derived from the MDM2 promoter (5,000 cpm; 1.0 ng of DNA
per assay). The reaction was incubated at room temperature for 30 min
and directly loaded onto a 4% nondenatured gel.
Western Blot Analysis--
Transfected cells were harvested for
preparation of nuclear extracts. Nuclear extracts containing 150 µg
of proteins were directly loaded onto an SDS gel, and proteins were
detected by ECL reagents (Bio-Rad) after Western blotting using
antibodies as indicated in the figure legends.
Transient Transfection and Luciferase Assay--
H1299 cells
(60% confluence in a 12-well plate) were transfected with a
pCMV- cDNA Probe Labeling--
p53 and p73 cDNA probes, as
indicated in Fig. 2B, were prepared using random primer
labeling as described (47).
Northern Blot Analysis--
Northern blot analysis was carried
out as described (47). Total RNA from transfected cells, as indicated
in Fig. 2B, was isolated using the Trizol reagent (Life
Technologies, Inc.). 15 µg of RNA were loaded onto a 1.5% agarose
gel and transferred to a nitrocellulose membrane. The membrane was
exposed to UV light in a UV cross-linker (Fisher Biotech) and incubated
with 32P-labeled cDNA probes encoding mouse p53 or p73
at 42 °C overnight. After washing with 4× SSC once and 1× SSC
twice, the blot was exposed to x-ray film.
p300 Does Not Acetylate the Full Length of p73 p300 Does Not Acetylate p73
Consistent with the published studies by others and us (13, 49), the
p53 level increased when p300 was cointroduced into the cells (compare
lane 7 with lane 8 of the top panel).
The acetylase activity is partially responsible for this increase, as
the p53 level was elevated to a lesser degree when the p300 mutant,
which did not acetylate p53, was cotransfected with p53 (lane
6). This result was reproduced in separate experiments (data not
shown). Similarly, the p73 p300 Joins and Stabilizes the p73-DNA Complex in
Vitro--
Because p300 enhances the ability of p53 to bind the
specific p53RE motif by acetylating this protein (11, 13), we tested whether p300 affects the sequence-specific DNA binding ability of
p73
As expected (11, 13), p300 (100 ng) markedly stimulated p53-DNA complex
formation in the presence of acetyl-CoA (lanes 3 and
4 of Fig. 3C). However, this acetylase only
caused a marginal increase of the p73 Stimulation of p73
We previously showed that p73 interacts with p300 through the N
terminus of p73 and the CH1 domain of p300 (15), and this interaction
mediates p73-dependent transcription and apoptosis (15).
The study presented here provides several lines of evidence showing
that p300 positively affects p73-mediated transcription activation
independently of its intrinsic acetylase activity. First of all, p73
How does p300 modulate p73 function without acetylating this protein?
One possibility would be that p300 may regulate p73 stability and thus
enhance its activity. The p73
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
irradiation, p73 is activated through the
c-Abl tyrosine kinase (29-31), whereas p53 is activated through the
Ataxia telangiectasia-mutated Chk2 pathway (32-36).
Also, MDM2 mediates degradation of p53, but not p73, although it
inhibits the functions of both the proteins (14, 37-42). In addition,
unlike p53, p73 contains a unique C-terminal SAM
(sterile alpha motif)-like
domain that may be involved in protein-protein interaction (43, 44).
Distinct mechanisms for the regulation of the p53 family members may,
at least partially, account for their different physiological roles in
development, homeostasis, or tumorigenesis. Thus, unraveling their
regulatory mechanisms is crucial for better understanding of their
biological roles.
in vitro. Also, p300 did not acetylate p73
in vivo when both of the proteins were overexpressed
in cells. Moreover, an acetylase-defective mutant p300 named p300AT2,
like wild type p300, was able to elevate p73
-dependent
transcription in cells. These results demonstrate that p300 does not
need its acetylase activity to stimulate p73-dependent transcription.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and
affinity-purified as described (46). This anti-p73 antibody recognizes
both p73
and p73
. Baculovirus harboring human p300 mutant AT2 and
pBlueScript-p300AT2 were generously provided by W. Lee Kraus (Cornell
University, Ithaca, NY) and James T. Kadonaga (University of
California, San Diego, CA). The p300AT2 insert with a C-terminal
polyhistidine tag was cloned into the HindIII and
NotI sites of pcDNA3 vector (Invitrogen).
,
and the p73
C Terminus (aa 311-636)--
p300AT2 and p300
were purified from baculovirus-infected SF9 insect cells using
immunoaffinity columns as described. His-p53, His-p73
, and the
p73
C terminus (aa 311-636) tagged with histidines were purified
from bacteria using nickel-nitrilotriacetic acid column as described
(14, 15).
-galactoside reporter plasmid (0.2 µg) and a luciferase
reporter plasmid (0.1 µg) driven by two copies of the p53RE motif
derived from the MDM2 promoter (14), together with a combination of
different plasmids (total plasmid DNA = 1 µg/well) as indicated
in Fig. 4, using LipofectAMINE (Promega, Madison, WI). 48 h
post-transfection, cells were harvested for luciferase assays as
described previously (15). Luciferase activity was normalized by a
factor of
-galactosidase activity tested in the same assay.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
in
Vitro--
The acetyltransferase activity of p300 has been shown to be
important in mediating the activity of the transcriptional activator p53 (11-13). To check whether this is also true for the p53 homolog p73
(16), we conducted in vitro acetylase assays using
recombinant p300 purified from baculovirus and his-p73
or his-C
terminus of p73
(aa 311-636) purified from bacteria, as well as
14C-acetyl-CoA as substrates. Surprisingly, as shown in
Fig. 1A, p300 was unable to
acetylate the intact p73
protein (lanes 3 and
4). This was not because of the inactivity of the purified p300 protein, as it was active in acetylating p53 (lane 1),
as well as the C-terminal domain of p73 (lanes 5 and
6). As shown in the representative result (Fig.
1B), even though up to 5 µM of acetyl-CoA was
used, p300 (200 ng) was still not able to acetylate p73
(lanes
1-7). Also, 1 µg of p300 did not show efficient acetylase activity on p73
(data not shown). In contrast, p300 needed only 100 nM of acetyl-CoA to sufficiently acetylate p53 (lane
11) and ~1 µM acetyl-CoA to reach a plateau level
(lanes 8-14). These results indicate that p300 does not
utilize the full-length p73
as a substrate in vitro.
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Fig. 1.
p300 does not acetylate
p73 in vitro. A,
p300 acetylated the C-terminal region aa 311-636 but not the full
length of p73
in vitro. 150 ng of p53, 150 or 300 ng of
p73
or of the p73
C-terminal region aa 311-636
(C-p73
), and 200 ng of p300 were used in this acetylation
assay, as indicate on top. 14C-Labeled
acetyl-CoA was used here. Acetylated proteins were detected by
autoradiography. Molecular mass markers are indicated at
left, and * denotes nonspecific signals (the same for Figs.
2-4). B, p300 acetylates p53, but not p73
, in
vitro in an acetyl-CoA concentration-dependent
fashion. 250 ng of p53 or p73
and 200 ng of p300 together with
different concentrations of nonlabeled acetyl-CoA, as indicated on top,
were used in this assay. Acetylated proteins (upper panel)
were detected by Western blot (WB) using antibodies specific
for acetylated lysines (Upstate Biotechnology, Inc.). The p53 or p73
protein (lower panel) was detected by WB using anti-p53 or
anti-p73 antibodies.
in Vivo--
Next, we tested
whether p300 acetylates p73
in vivo. To this end, human
p53 null lung small cell carcinoma H1299 cells were transiently
transfected with plasmids encoding wild type, mutant p300 (p300AT2)
lacking acetylase activity (48), p53, or p73
, as indicated in Fig.
2A. Acetylation of p53 was
detected by Western blot using polyclonal antibodies specifically
against acetylated lysines. As expected, p300 but not the p300AT2
mutant acetylated p53 in cells (lanes 6 and 7 of
the bottom panel). By striking contrast, p73
acetylation
was not detectable in the presence of wild type p300 (lane
2), despite the fact that the p73
protein was well expressed
(top panel), and the transfected cells were exposed to UV or
irradiation (data not shown). This was not because of the inability
of the anti-acetylated lysine antibody to recognize the acetylated
p73
, as this same antibody was able to immunoreact with the p73
C-terminal region that was acetylated by p300 in vitro (Ref.
13 and data not shown). These results clearly show that p300 does not
acetylate p73
in cells.
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Fig. 2.
p300 does not acetylate
p73 in vivo. A,
p300 acetylated p53 but not p73
in cells. H1299 cells
(105 cells/60-mm dish) were transfected with p73
(500 ng) or p53 (500 ng) alone or together wild type (WT) or
mutant (AT2) p300 (2.5 µg), as indicated on top. Whole
cell extracts (500 µg) of the transfected cells were directly loaded
onto an 8% SDS gel. As indicated on left, p73
and p53
were detected by WB using the monoclonal antibodies against p73 and p53
(top panel). The acetylated proteins were detected by WB
using polyclonal antibodies against acetylated lysines (bottom
panel). Lane 5 denotes the molecular mass markers, and
the acetylated bovine serum albumen was detected by the anti-acetylated
lysine antibody (bottom panel). B, overexpression
of p300 did not influence the mRNA synthesis of either p73
or
p53 in cells. Northern blot analysis (NB) of the mRNA
levels of p73
and p53 after the same transient transfection as
described in panel A is shown. Plasmids used are
indicated on top of both panels. 13 µg of total
RNAs isolated from the transfected cells were loaded onto a 1% agarose
gel. Ethidium bromide staining for total RNA loading was shown on the
lower panel. The mRNA for either p73
or p73 was
detected by NB using the 32P-labeled p73
or p53 probes
(upper panel).
level also increased when cotransfected
with p300 (lane 2). However, this was not dependent upon the
acetylase activity of p300, as p300 did not acetylate p73
(compare
lane 2 with lane 3 of the bottom
panel), and overexpression of the acetylase-dead mutant p300AT2
also increased the level of p73 (lane 1). Because the
mRNA synthesis of the exogenous p53 or p73
was not affected by
the exogenous p300 (Fig. 2B), the increase of either the p53
or p73
protein was not due to transcriptional activation of these
exogenous genes by p300. These results, which were reproducible,
suggest that although p300 did not acetylate p73
, it may modulate
the stability of this transcriptional activator.
without acetylating this protein. First, we established a gel
mobility shift assay, using the recombinant p73
protein purified
from bacteria and the radiolabeled DNA oligomer probes containing the
p53RE sequence derived from the p21 promoter (Fig. 3A). The p73
-DNA complex
migrated slower than the p53-DNA complex and was specifically inhibited
by the nonlabeled p53RE-containing oligomers but not dIdC. Also,
this DNA-protein complex was supershifted by antibodies specifically
against p73 but not p63 (Fig. 3B).
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Fig. 3.
p300 binds to the p53RE-bound
p73 but does not enhance the
p73
-DNA complex formation in
vitro. A, the formation of p73
-DNA
complexes was inhibited by the p53RE-containing oligomers. 100 ng of
p53 or p73
, 100 (1×) or 200 ng (2×) of
poly (dI-dC) or the p53RE-containing oligomers derived from the
p21 promoter (p21) were used in this EMSA experiment, as indicated on
top. B, the p73
-DNA complexes were
supershifted by the monoclonal anti-p73 antibody. The same assay was
conducted as above, except 100 (1×) or 200 ng
(2×) of the anti-p63 or anti-p73 antibody were used here,
as indicated on top. C, p300 bound to the
p73
-DNA complexes. The same EMSA experiment was carried out as
above, except 100 (1×), 200 (2×), or 300 ng
(3×) of p300 purified from the baculovirus system, either
alone or with the anti-p300 antibody (200 ng), were pre-incubated with
the p73
protein before being added into the reaction, as indicated
on top. In addition, 5 µM of nonlabeled
acetyl-CoA were added into lanes 2, 4, and
6-11. Free probes are shown at the bottom of
each panel.
-DNA complex, even when 2-fold
more p300 (200 ng) was used (lanes 5-11). This slight
effect was not reproduced (data not shown). Rather, p300 participated
in the p73-DNA complex, because addition of increasing amounts of p300
supershifted this complex (lanes 6-7), and this complex was
further supershifted by the anti-p300 antibody (lanes
9-11). By participating in the p73-DNA complex, p300 may
stabilize this complex, because the amount of the complex increased
apparently when 300 ng of p300 was added into the reaction (lanes
8 and 11). This increase was also seen in the absence
of acetyl-CoA (data not shown). These results suggest that p300 joins
and stabilizes the p73-DNA complex through direct protein interaction
but not through its acetylase activity.
-dependent Transcription by the
Acetylase-defective Mutant p300--
To further test whether the
acetylase-defective p300AT2 mutant is able to influence p73
function
in cells, H1299 cells were transfected with the p73
expression
plasmid alone or together with plasmids encoding wild type p300 and its
two mutants, p300AT2 and the deletion lacking the central portion from
aa 242 to aa 1740 (
242-1740), in the presence of the p53RE-driven
luciferase reporter plasmid. Luciferase activity was measured as an
indication of p73
-dependent transcription (Fig.
4C). We previously showed that
242-1740 did not bind to p73 and had no effect on its transcription activity (15). Consistently, this mutant did not increase the level of
p73
(two upper panels of Fig. 4B), and thus,
was used here as a negative control. As expected, this mutant neither
acetylated p53 nor increased its protein level (two lower
panels of Fig. 4B). In agreement with our previous
study (15), p300 markedly stimulated p73
-dependent
transcription, whereas the deletion mutant had no effect on this
transcription activity (Fig. 4C). The same was true for p53
(data not shown; see Ref. 11). However, the acetylase-defective p300AT2
mutant, like wild type p300, enhanced p73
-mediated transcription
markedly. This enhancement would not be due to the general effect on
nucleosome modeling or modification, because this mutant was inactive
in acetylating histones (Fig. 4A) and did not affect the
mRNA level of p73
(Fig. 2B). These results indicate
that p300 can stimulate p73
-mediated transcription without involving
its intrinsic acetylase activity.
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Fig. 4.
Both of the wild type and mutant p300
proteins stimulate p73 -dependent
transcription in cells. A, the p300 mutant (p300AT2)
was inactive in acetylating p53 and histones in vitro. An
in vitro acetylation assay was performed as described in
Fig. 1B. 100 ng of p53 or 400 ng of histones (Sigma)
together with 1 µM acetyl-CoA were used in this assay.
100 or 200 ng of wild type p300 or p300 mutant (AT2) were used here, as
indicated on top. Acetylated proteins were detected by WB
using anti-acetylated lysine antibodies. p300 was detected using the
polyclonal anti-p300 antibody (top panel). B, the
242-1740 mutant of p300 did not stimulate the levels of p73
and
p53. The same transfection-Western blot assay as that described for
Fig. 2A was carried out, except that
pcDNA3-
242-1740, instead of p300AT2 (2.5 µg), was used
together with either p73
or p53 as indicated on top.
Antibodies used for WB are shown at left. The level of p73
or its acetylation is shown on two top panels from one
experiment (Exp 1), whereas that of p53 is shown on
two bottom panels from a separate experiment (Exp
2). C, transient transfection-luciferase assays. H1299
cells (60% confluence in 35-mm dishes) were transfected with 100 ng of
p73
alone or with 600 ng of p300, p300AT2 or the p300 deletion
mutant without the region of aa 242-1720, 200 ng of
-galactosidase
reporter (as an internal control), and 100 ng of the luciferase
reporter plasmid driven by the p53RE motif derived from the p21
promoter. Each column presents the mean activity from three
dishes, and the bars denote the deviation errors. The
pcDNA3 parental plasmid was used as a control (c).
Luciferase activity was normalized with
-galactosidase
activity.
was a considerably poor substrate for the acetylase activity of p300
in vitro, in contrast with p53, which is acetylated by p300
in response to DNA damage (12, 13). Consistent with this in
vitro result, p300 did not acetylate p73
when both of the
proteins were overexpressed in cells. p73
was also not acetylated by
p300.2 Moreover, the
acetylase-defective mutant p300AT2 was able to stimulate
p73-dependent transcription in transient transfected cells.
Thus, these results demonstrate that p300 does not need its acetylase
activity to activate p73 function. Although our study does not exclude
the possibility that p73
may be regulated through acetylation by
other acetylases such as CBP or p300/CBP-associated factor (12,
50, 51), this appears less likely, because we were not able to detect
acetylated p73 molecules by Western blot using antibodies against the
acetylated lysine, even when this protein was overexpressed, or the
transfected cells were irradiated with UV or
ray (Fig. 2 and data
not shown).
protein level increased when
coexpressed with p300 (Fig. 2 and Fig. 4B). However, the
mRNA level of p73
was not affected by p300 (Fig. 2B),
suggesting that p73
is regulated at the protein level and probably
stabilized by p300. Alternatively, p300 may serve as a bridging protein
that links p73 with the RNA polymerase II transcriptional machinery. Supporting this is that p300 interacts with p73 when this
transcriptional activator binds to its responsive DNA element sequence
(Fig. 3C). Although these two possibilities may coexist,
further investigation of these questions is necessary to elucidate the
detailed molecular and biochemical mechanism of p73 regulation by p300.
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ACKNOWLEDGEMENTS |
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We thank David Keller for active discussion, Eric Kobet for preparing reagents for this study, and W. Lee Kraus, James T. Kadonaga, and Richard Goodman for generously providing reagents used in this study.
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FOOTNOTES |
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* This work was supported by National Institutes of Health and American Cancer Society grants (to H. L.).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.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Oregon Health Science University, 3181 SW Sam Jackson Park Rd., Portland, OR 97201. Tel.: 503-494-7414; Fax: 503-494-8393; E-mail: LUH@OHSU.edu.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.C000722200
2 Unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: CBP, cAMP-response element-binding protein-binding protein; EMSA, electrophoresis mobility shift assay; NB, Northern blot; RE, responsive element; WB, Western blot; aa, amino acid; MDM, murine double minute.
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