From the Department of Pathology, Harvard Medical School and § Department of Radiation Biology, Harvard School of Public Health, Boston, Massachusetts 02115
Received for publication, September 5, 2000, and in revised form, January 29, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
p300 and CREB-binding protein (CBP) are related
transcriptional coactivators that possess histone acetyltransferase
activity. Inactivation of p300/CBP is part of the mechanism by which
adenovirus E1A induces oncogenic transformation of cells. Recently, the
importance of p300/CBP has been demonstrated directly in several
organisms including mouse, Drosophila, and
Caenorhabditis elegans where p300/CBP play an indispensable
role in differentiation, in patterning, and in cell fate determination
and proliferation during development. CBP/p300s are modified by
phosphorylation during F9 cell differentiation as well as adenovirus
infection, suggesting that phosphorylation may play a role in the
regulation of p300/CBP activity. Here we show that the
mitogen-activated/extracellular response kinase kinase 1 (MEKK1)
enhances p300-mediated transcription. We identify several domains
within p300 that can respond to MEKK1-induced transcriptional
activation. Interestingly, activation of p300-mediated transcription by
MEKK1 does not appear to require the downstream kinase JNK and may
involve either a direct phosphorylation of p300 by MEKK1 or by other
non-JNK MEKK1-directed downstream kinases. Finally, we present evidence
that p300 is important for MEKK1 to induce apoptosis. Taken together,
these results identify MEKK1 as a kinase that is likely to be involved
in the regulation of the transactivation potential of p300 and support
a role of p300 in MEKK1-induced apoptosis.
p300 was initially identified as an adenovirus E1A-associated
cellular protein in coimmunoprecipitation experiments (1, 2). The
ability of adenovirus E1A to immortalize cells and to induce full
morphological transformation is dependent on its ability to interfere
with the functions of p300 and its related protein
CBP1 (3-5) and members of
the retinoblastoma family members (6-9). In addition, inactivation of
p300 and CBP is necessary for E1A to inhibit differentiation (10-12).
These experiments suggest that p300/CBP play an important role in cell
proliferation and differentiation. Recent experiments in mouse,
Drosophila, and Caenorhabditis elegans have
provided direct evidence that p300/CBP are crucial in both differentiation and cell proliferation during development (13-15).
p300 and CBP are both transcriptional cofactors that can acetylate
histones and transcription factors due to their histone acetyltransferase (HAT) activity (16-19). This suggests that p300 and
CBP may regulate transcription in part by modifying the chromatin structure as well as activities of transcription factors (18-20). Consistent with this, a C. elegans homolog, CBP-1, has been
shown to promote endoderm differentiation by antagonizing the histone deacetylase activity, highlighting the importance of the biological significance of the HAT activity of CBP-1 (13). Additional mechanisms that account for p300/CBP-mediated transcriptional activation involve
direct interactions with the basal factors (4, 21, 22) as well as with
the RNA polymerase II complex (23).
Although a great deal is known about p300/CBP as transcriptional
regulators, very little information is available about mechanisms that
regulate their transcriptional activity. Several lines of evidence
suggest that phosphorylation may regulate p300/CBP activity. First,
p300/CBP have been shown to be phosphoproteins whose phosphorylation state alters during F9 cell differentiation and in adenovirus infection
(24). Second, kinases such as protein kinase A and mitogen-activated
kinases have been shown to activate CBP-mediated transcription (25,
26). Third, p300 has been reported to be phosphorylated in
vitro by cyclin-dependent kinases such as cdc2 and
cdk2 (27). In this report, we provide evidence that the mitogen-activated protein kinase/ERK kinase kinase 1 (MEKK1) stimulates p300-mediated transcription in vivo in a manner that is
independent of its downstream kinase JNK.
MEKK1 is a 196-kDa protein which, when cleaved by caspases in response
to genotoxic agents, is capable of inducing apoptosis (28, 29). MEKK1
propagates the signal by activating its downstream kinase, MKK4, which
in turn activates JNK (30, 31). Activated JNK translocates into the
nucleus where it modifies the activity of transcription factors such as
AP-1 (32, 33). MEKK1 has also been shown to activate NF Cell Culture--
HeLa, U2OS, and MCF-7 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 2 mM L-glutamine, and antibiotics.
Plasmids--
pGAL4-p300 aa 1-596, pGAL4-p300 aa 744-1571, and
pGAL4-p300 aa 1257-2414 were described before (22). pGAL4-Sp1Q2 was
obtained from Robert Tjian (University of California, Berkeley).
p12SE1A expression plasmid was described previously (37). pGAL4-p300 aa
19-2414, pGAL4-p300 aa 1737-2414, and pGAL4-p300 aa 1945-2414 were
kindly provided by Antonio Giordano (Jefferson Cancer Institute, Philadelphia, PA). pCDNA3-FLAG MEKK1 Luciferase Assay--
HeLa cells were transfected using Fugene 6 transfection reagent (Roche Molecular Biochemicals) with indicated
reporters and cDNA expression vectors. After 40 h, luciferase
activity was determined as described (38). The luciferase activities
were normalized on the basis of Assay for the Effect of Nocodazole on p300-dependent
Transcription--
U2OS cells were transfected with mammalian
expression plasmids encoding either GAL4 DNA-binding domain (G4, 1 µg) alone or G4-p300 (1 µg) together with the target reporter
G4-luciferase (0.5 µg). One hour before harvest, the cells were
treated with two different doses (0.5 and 1.0 µg/ml) of nocodazole.
Cell extracts were prepared and used for luciferase assay described above.
Expression and Purification of Recombinant
Proteins--
GST-p300 fusion proteins were induced and purified as
described (34). His6 MEKK1 In Vitro Kinase and Apoptosis Assays--
About 5 µg of
purified GST fusion proteins were suspended in 30 µl of kinase buffer
(20 mM MOPS, pH 7.2, 2 mM EDTA, 10 mM MgCl2, 0.1% Triton X-100) and incubated
with either 0.5 µg of His6 MEKK1
Nocodazole stimulation of MEKK1 kinase activity was measured by the
activation of JNK. Briefly, proliferating U20S cells were treated with
either Me2SO solvent control or nocodazole at the indicated concentrations. Cell lysates were prepared 1 h
post-treatment, and the activation of MEKK1 was analyzed by Western
blotting with an anti-JNK1 antibody (SC-474, Santa Cruz Biotechnology)
as described previously (39).
Apoptosis assays were performed using MCF-7 cells that express either
an active p300 or inactive p300 ribozymes as described previously (40).
Activated MEKK1 was cotransfected into MCF-7 cells along with green
fluorescent protein (GFP) cDNA. Apoptotic cells were scored on the
basis of GFP and Hoecht stain 48 h after transfection.
Cell Fixation and Immunofluorescence Microscopy--
U2OS cells
were transiently transfected with full-length HA-MEKK1 or FLAG-MEKK1 MEKK1 Nocodazole Stimulates p300-dependent
Transcription--
To determine whether extracellular stimuli known to
activate MEKK1 can stimulate p300-dependent transcription,
we examined the ability of nocodazole (35, 36) to regulate
GAL4-p300-mediated transcription. As shown in Fig.
2A, MEKK1 kinase activity is
stimulated by nocodazole in a dose-dependent manner as
shown by its ability to induce JNK phosphorylation (compare lanes
2 and 3 with lane 1). When the kinase
activity of MEKK1 is stimulated, we find consistent enhancement of
GAL4-p300-mediated transcription by nocodazole in a
dose-dependent manner, although at a modest level (Fig.
2B, lanes 5 and 6). Taken together, activated
MEKK1 provided either exogenously (transfection) or endogenously
(conversion of an inactive MEKK1 to an active one by nocodazole) can
stimulate GAL4-p300-mediated transcription.
Identification of Domains of p300 Involved in Its Response to
MEKK1--
Previously, we and others (21, 22) have shown that at least
two independent activation domains are present in the N- and C-terminal
portions of p300. We wished to determine whether the transcriptional
activity of these two p300 domains could be regulated by MEKK1
We next sought to identify a minimal domain within the N-terminal and
C-terminal regions of p300 that were responsible for the
transcriptional activation by MEKK1
The C-terminal deletion mutants of p300 were also analyzed for the same
purpose. Fig. 3 shows that MEKK1 MEKK1 Stimulation of GAL4-p300-mediated Transcription Does Not
Require JNK--
It has been well documented that MEKK1 activates the
JNK family members of kinases (30, 31, 44). To determine whether JNK1
was involved in the activation of GAL4-p300-mediated transcription induced by MEKK1, we first asked whether JNK is necessary for the MEKK1
response mediated by the subdomains of p300 described above. We tested
a dominant negative inhibitor of JNK1 (JNK1 APF) (32) for its ability
to block MEKK1
The above results predict that these subdomains of p300 are either not
substrates for JNK1, or the JNK phosphorylation sites residing in these
subdomains are not important for MEKK1-induced, p300-mediated
transcription. To determine whether JNK1 can phosphorylate the
MEKK1-responsive p300 subdomains, we performed in vitro
kinase assays using the various p300 domains fused to GST as substrates for recombinant JNK. Equal amounts of GST proteins were used for the
in vitro kinase assays (data not shown). Fig.
5 shows that recombinant JNK
phosphorylates GST-p300 aa 1709-1913 (C3) very strongly (lane
4). In contrast, recombinant JNK barely phosphorylated GST-p300 aa
2-337 or GST-p300 aa 302-667 (Fig. 5, lanes 2 and 3). Similar results were found in JNK immune complex kinase
assays using the same GST-p300 substrates (data not shown). These
results indicate that only the C3 domain of p300 was a potential
substrate for JNK, whereas the N-terminal domains were not. The fact
that the N-terminal domains of p300 are poor JNK substrates is
consistent with the above finding (Fig. 5) that JNK is not involved in
MEKK1-induced transcription mediated by the N-terminal domains of
p300.
The finding that JNK phosphorylates GST-p300 aa 1709-1913, which can
also mediate a MEKK1 response, appears to be contradictory to the
observation that the p300 C-terminal domain-mediated MEKK1 response is
unaffected by a dominant negative form of JNK (Fig. 4). To resolve this
issue, we determined whether JNK phosphorylation of this domain is
correlated with its ability to mediate a MEKK1 response. We therefore
mutated all potential JNK sites with the consensus sequence
serine/proline or threonine/proline within this domain. Because of the
large number of proline-directed serines and threonines within this
region, we made a series of cluster mutations designated
A-D shown in Fig.
6A. Equal amounts of the mutant GST fusion proteins (Fig. 6C) were then tested for
their ability to be phosphorylated by recombinant JNK. Fig.
6B shows that compared with wild-type p300 aa 1709-1913,
mutation of proline-directed serines and threonines to alanines within
either clusters A or B partially abolished the ability of recombinant
JNK to phosphorylate GST-p300 aa 1709-1913 (lanes 2 and
3). Mutation of serines and threonines to alanines within
clusters C or D did not inhibit the ability of JNK to phosphorylate
GST-p300 aa 1709-1913 (data not shown). However, mutant GST-p300 aa
1709-1913, which combines the mutations in both clusters A and B
(mut A/B), totally lost the ability to respond to JNK-induced
phosphorylation (Fig. 6B, lane 4).
We next analyzed the ability of this JNK phosphorylation-defective
mutant for its ability to mediate an MEKK1 response. As shown in Fig.
6D, mutation of all potential JNK sites (clusters A-D) did not inhibit the response of this GAL4-p300 aa 1709-1913 mutant to MEKK1 MEKK1 Can Phosphorylate the N Terminus of p300 in Vitro--
Given
the fact that JNK1 is not involved in the response of p300 to MEKK1,
nor are the other potential downstream kinases such as p38 and
I Analyses of Subcellular Localization of MEKK1 and p300--
The
model that MEKK1 may regulate p300 activity by directly phosphorylating
p300 predicts that the active form of MEKK1 resides in the nucleus, and
these two proteins may physically interact. To address this question,
we analyzed the localization of endogenous p300 in the presence and
absence of either active or inactivate MEKK1. As expected, we find
MEKK1
Inactive MEKK1 resides predominantly in the cytoplasm (see
Ref. 29 and Fig. 8A). As shown in Fig. 8A,
HA-tagged full-length MEKK1 (inactive form) is present in both diffused
and punctate patterns in the cytoplasm (Fig. 8A). On the
other hand, endogenous p300 is present in the nucleus (see Ref. 12, and
Fig. 8B, open arrow, indicates an untransfected
cell in which p300 is present in the nucleus). Interestingly and
strikingly, in the cells that overexpress MEKK1 (Fig. 8,
arrowheads), p300 staining shows a significant alteration.
In addition to the nuclear staining, it is also present in a punctate
pattern in the cytoplasm, identical to the punctate HA-MEKK1 pattern
(Fig. 8B, HA- MEKK1-transfected cell indicated by a
closed arrow). This suggests that some of the p300 molecules
may have been retained by HA-MEKK1 in the cytoplasm, perhaps through
protein-protein interactions. Taken together, analyses of the
subcellular localization of p300 as well as MEKK1 (both active and
inactive forms) lend further support to the idea that MEKK1 can
regulate p300 activity directly, independent of its downstream JNK kinase.
MEKK1-induced Apoptosis Is Inhibited in a p300-active Ribozyme Cell
Line--
It has been documented that MEKK1 activation can induce
cells to undergo apoptosis (29, 45, 46). To determine whether p300 is
involved in the MEKK1 apoptotic pathway, we made use of an MCF-7 cell
line that expresses a much reduced level of p300 due to the presence of
an active p300 ribozyme (40). As shown previously, these cells, but not
MCF-7 cells containing an inactive p300 ribozyme, are deficient in the
apoptotic response of cells to ionizing radiation (40). We asked if
activated MEKK1 induces apoptosis in MCF-7 cells in a manner that may
be dependent on p300. As shown in Fig. 9,
compared with the inactive p300 ribozyme cell line which contains
wild-type level of p300, the active p300 ribozyme cell line showed a
50% reduction in apoptosis induced by MEKK1 p300/CBP participate in the integration of a diverse array of
signals with transcriptional events that govern gene expression. In
this report, we demonstrate that a kinase in the mitogen-activated kinase pathway, MEKK1, can robustly enhance transcription mediated by
p300 when fused to the GAL4 DNA binding domain. This is likely to be
physiologically relevant as nocodazole, an extracellular stimulus of
MEKK1, can also enhance p300-dependent transcription. MEKK1
has been shown to be important for the apoptotic response of cells to
ionizing radiation (29). Significantly, the dynamic interaction between
MEKK1 and p300 may be important for MEKK1-induced apoptosis.
MEKK1 has been shown to activate the JNK family of the
mitogen-activated kinase members by activating MKK4 (30, 31, 44), an
immediate upstream activator of JNK. Surprisingly, our results show
that JNK is not involved in the activation of
p300-dependent transcription. First, dominant negative JNK1
does not block MEKK1-induced activation of GAL4-p300-mediated
transcription. Second, mutations of all potential JNK sites
(proline-directed serines or threonines) within the N2 or C3 region do
not impair the ability of MEKK1 to enhance p300-mediated transcription.
Since MEKK1 can phosphorylate p300 in vitro, these results
taken together raise the possibility that MEKK1 may induce
p300-mediated transcription by directly phosphorylating p300,
particularly within the p300 N2 region. However, since p300 N1 and p300
C3 regions are only weakly phosphorylated by MEKK1 itself (Fig.
7A), it is possible that the ability of MEKK1 to activate
GAL4-p300-mediated transcription may also involve additional
unidentified MEKK1 downstream kinases. Taken together, our data suggest
that stimulation of p300-mediated transcription by MEKK1 may occur via
both direct and indirect mechanisms.
Since p300 is a large platform protein and can interact with many
different transcription factors, our results cannot rule out the
possibility that the effect of MEKK1 on p300 may be mediated by
modifications of the interacting transcription factors. For instance,
p300 interacts with NF The ability of MEKK1 to regulate transcription directly is not
unprecedented. MEKK1 has been shown to increase the transcriptional activity of GAL4-c-myc and GAL4-Elk-1 (46). The activation of GAL4-c-myc-dependent transcription is believed to also
occur in a JNK-independent manner (46). Together with the results
described here, these findings suggest that direct phosphorylation of
transcriptional regulators by MEKK1 may be a common mechanism by which
MEKK1 regulates transcription. Consistent with this idea, we find the
active form of MEKK1 (MEKK1 At present, the phosphoacceptor sites on p300 targeted by MEKK1 or its
unknown downstream kinases are not defined. We have mutated all
potential JNK sites that consist of proline-directed serines and
threonines within p300 aa 302-667 and aa 1709-1913 without causing
alterations in the response to MEKK1. In addition, mutations of serines
in a putative MEKK1 phosphorylation motif (DSXXXS) (48)
located within p300 aa 302-667 did not abolish the ability of this
region to respond to MEKK1 (data not shown). Further studies are
necessary to identify the amino acid residues that are either directly
or indirectly phosphorylated by MEKK1.
How does phosphorylation of p300 leads to an enhancement of its
transcriptional activity? p300 has been shown to interact with members
of the basal transcriptional machinery such as TFIIB and TATA-binding
protein (4, 21, 22, 26) as well as the RNA polymerase II complex (23).
Perhaps one mechanism by which MEKK1 increases the transactivation
potential of p300 is to promote the interaction of p300 with these
members of the transcriptional basal machinery. An alternative but not
mutually exclusive possibility is that phosphorylation of p300 by MEKK1
may enhance the HAT activity of p300. It should be interesting in the
future to test directly these possibilities.
Activated MEKK1 has been shown to induce cell death (28, 45, 46, 49).
We provide evidence here that p300 may be important in mediating MEKK1
induction of the apoptotic pathway in MCF-7 cells. Impaired expression
of p300 in MCF-7 cells resulted in significantly lower levels of
apoptosis induced by MEKK1 (Fig. 9). As discussed above, the regulation
of p300 activity by MEKK1 does not require its downstream kinase JNK.
Interestingly, the MCF-7 cell death response to MEKK1 also appears to
be JNK-independent (29, 46). Collectively, these findings suggest that
the ability of MEKK1 to induce apoptosis in MCF-7 cells may involve an
increase in the transcriptional activity of p300.
MEKK1-induced apoptosis appears to involve stabilization of p53 as well
as an increase in its transcriptional activity (50). Given the fact
that p300 is a coactivator for p53 (41, 51, 52), it is possible that
MEKK1-induced phosphorylation of p300 may be important for p300 to
serve as a coactivator for p53. It is interesting to note that p300
appears to be essential for the stabilization of p53 as well as
increase in p53-dependent transcription in response to
ionizing radiation (40). Therefore, it is also possible that
phosphorylation of p300 by MEKK1 may contribute to the interaction of
p300 with p53 and its stabilization, resulting in changes of
p53-dependent gene expression and apoptosis in MCF-7 cells.
In summary, our data show that MEKK1 can significantly enhance
transcription mediated by p300. We have identified several regions
within p300 that are responsive to MEKK1. The MEKK1-induced transcription mediated by p300 does not appear to require its known
downstream kinase JNK. Instead, regulation of p300 activity by MEKK1
may occur via a direct phosphorylation of p300 by MEKK1 and by other
currently unknown MEKK1 downstream kinases. Biologically, impaired
expression of p300 in MCF-7 cells significantly inhibits the amount of
apoptosis induced by MEKK1
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B- and
c-Myc-mediated transcription, the former involving the activation of
the I
B
kinase (34). We find that the activated MEKK1 can
significantly increase GAL4-p300-mediated transcription. Consistently,
nocodazole, which is an extracellular stimulus of MEKK1 (35, 36), can
also stimulate p300-dependent transcription. Interestingly,
this activation appears to be independent of the downstream kinase JNK
since a dominant negative JNK fails to abrogate MEKK1-induced,
p300-mediated transcription. In addition, mutations of all the possible
JNK sites within subdomains of p300 responsive to MEKK1 had no effect
on their response to MEKK1. Although the mechanisms by which MEKK1
induces p300-mediated transcription are still unclear, one possible
scenario is a direct phosphorylation of p300 by MEKK1. Consistent with
this possibility, we find that MEKK1 can robustly phosphorylate p300
directly in vitro. Significantly, we also find that the
activated form of MEKK1 is present exclusively in the nucleus,
overlapping with the endogenous p300. In addition, we find the
transfected inactive MEKK1 in the cytoplasm, and it appears to localize
some of the p300 proteins to the punctate MEKK1 signals. Finally, our
results also show that MEKK1-induced apoptosis is impaired by the
inhibition of p300 expression. Taken together, our results suggest that
phosphorylation of p300 induced by MEKK1 modifies its transcriptional
activity, which may be an important event in the apoptosis induced by
MEKK1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and pCDNA3-FLAG
MEKK1
(K432M), consisting of the C-terminal 321 residues of
full-length MEKK1, were obtained from Tom Maniatis (Harvard University,
Boston). pCDNA3 HA-tagged JNK (APF) was obtained from Tilang Deng
(University of Florida, Gainesville). GAL4-cJun aa 1-223 and
GAL4-luciferase were gifts from Michael Karin (University of
California, San Diego). GST-p300 aa 1709-1913 was provided by David
Livingston (Dana Farber Cancer Institute, Boston). pGAL4-p300 aa
302-667 was constructed by subcloning the region in frame with PSG 424 GAL4 expression vector. GST-p300 aa 2-337 and GST-p300 aa 302-667
were made by subcloning the corresponding regions into a compatible
pGEX vector (Amersham Pharmacia Biotech) to create an in-frame fusion.
GAL4-p300 aa 302-667 Ala-317/499/512/524/594 and GAL4-p300 aa
1709-1913 Ala-1726/1849/1851/1854/1857/1865/1868/1878/1906/1909 were
created by mutation of serines and threonines to alanines using the
QuikChange site-directed mutagenesis kit (Stratagene). Mutations were
verified by sequencing. GST-p300 alanine mutants were constructed by
cloning the regions into a compatible pGEX vector (Amersham Pharmacia Biotech) to create an in-frame fusion.
-galactosidase activity of
cotransfected Rous sarcoma virus
-galactosidase vector.
was purified from
baculovirus-infected Sf9 cell lysates using
nickel-nitrilotriacetic acid-agarose as described previously (34).
or 1 µg of
recombinant JNK (Stratagene) and 10 µCi of [
-32P]ATP
at 37 °C for 30 min. The kinase reactions were terminated by the
addition of Laemmli sample buffer. Proteins were resolved using 12%
SDS-PAGE gels and dried for autoradiography.
cDNA. Thirty six hours post-transfection, the cells were fixed in
3.7% paraformaldehyde at 25 °C for 10 min. After 5 min of washing
in PBS, the fixed cells were permeabilized in 0.2% Triton X- 100 (25 °C for 15 min) followed by 5 min of washing in PBS. The cells
were then incubated with primary antibody for 1 h at 25 °C.
Anti-HA epitope tag monoclonal antibody (Babco) and anti-FLAG epitope
tag monoclonal antibody (Sigma) were used at a 1:200 dilution;
anti-p300 polyclonal antibody C-20 (Santa Cruz Biotechnology) was
diluted 1:100. After three 10-min PBS washes, coverslips were incubated
in secondary antibody for 1 h at 25 °C. Fluorescein
isothiocyanate-conjugated goat anti-mouse (Jackson ImmunoResearch) and
Cy3 Red-conjugated goat anti-rabbit (Jackson ImmunoResearch) secondary
antibody were used at a dilution of 1:400. For the DAPI staining, the
coverslips were also incubated in 1× PBS with 1:10,000 dilution of
DAPI for 10 min. Vectashield 1200 media was used for slide mounting
(Vector Labs). Epifluorescence microscopy was conducted using a Nikon
microscope. Images were acquired by a Sony camera and analyzed using
Adobe Photoshop version 5.0.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Activates GAL4-p300-mediated Transcription--
To
determine whether MEKK1 affects p300-mediated transcription, a
constitutively activated MEKK1 mutant, MEKK1
(34), was cotransfected
into HeLa cells along with GAL4-p300 and a GAL4 luciferase reporter
construct. Fig. 1A shows that
MEKK1
augmented GAL4-p300-mediated transcription about 20-fold
compared with vector control. In contrast, a catalytically inactive
mutant of MEKK1, MEKK1
K432M (lysine 432 converted to methionine),
had little effect on GAL4-p300-mediated transcription (Fig.
1B). This indicates that stimulation of p300-mediated
transcription by MEKK1 is dependent on the kinase activity of MEKK1.
Furthermore, MEKK1
did not affect transcription mediated by
GAL4-Sp1Q2 (Fig. 1A), a previously characterized glutamine-rich activation domain of Sp1, suggesting that the ability of
MEKK1
to up-regulate p300-mediated transcription was not through a
general enhancement of the basal transcription machinery. The ability
of MEKK1
to activate GAL4-p300-mediated transcription was not
restricted to HeLa cells as similar results were obtained in COS-7
cells (data not shown). These findings suggest that a kinase pathway
involving MEKK1 can regulate GAL4-p300-mediated transcription.
View larger version (14K):
[in a new window]
Fig. 1.
Response of GAL4-p300 to activated
MEKK1. A, GAL4-p300 or GAL4-Sp1Q2 (1 µg) was
cotransfected into HeLa cells along with a GAL4-luciferase reporter in
the presence or absence of activated MEKK1 (MEKK1 ). Luciferase
activity was assayed after 48 h. Results, representative of three
experiments, describe the mean ± S.D. Luciferase activity in the
absence of MEKK1
was normalized to one. B, same as
A except that catalytically inactive MEKK1 (MEKK1
K432M)
was cotransfected into HeLa cells instead of activated MEKK1
.
View larger version (21K):
[in a new window]
Fig. 2.
Nocodazole stimulates GAL4-p300-mediated
transcription. A, nocodazole stimulation of MEKK1
kinase activity as measured by the activation of JNK. Proliferating
U20S cells were treated with either the solvent
Me2SO (lane 1) or nocodazole at 0.5 µg/ml (lane 2) or 1.0 µg/ml (lane 3). Cell
lysates were prepared 1 h post-treatment and analyzed by Western
blotting with an anti-JNK1. The JNK1 and phosphorylated JNK1 are
indicated by arrows on the right and labeled as
such. The concentration of nocodazole used is indicated. B,
nocodazole stimulates GAL4-p300-mediated transcription. Cells were
transfected as G4-Luc reporter plasmid together with either the GAL4
(lanes 1-3) or GAL4-p300 (lanes 4-6) expression
plasmids and were treated with either Me2SO
(lanes 1 and 4) or nocodazole (lanes 2 and 3, and 5 and 6) 1 h before
harvest. The y axis represents luciferase activity. The data
represent means ± S.D. of triplicates from two independent
experiments. The concentration of nocodazole used is indicated.
. Fig.
3 shows that the transcriptional activity
of the N-terminal (aa 1-596) and the C-terminal regions (aa
1257-2414), when fused to GAL4, were highly responsive to MEKK1
. In
contrast, the middle region of p300 (aa 744-1571), which itself has no
detectable transcriptional activity (22), did not respond at all to
MEKK1
.
View larger version (15K):
[in a new window]
Fig. 3.
Mapping of the p300 domain that responds to
activated MEKK1 . Diagram shows p300 and
its deletion mutants fused to the GAL4 DNA binding domain. The
black boxes indicate either the bromodomain (B)
or the cysteine/histidine (C/H) rich regions. The gray
box indicates the GAL4 DNA binding domain at the N terminus. HeLa
cells were transfected with GAL4-luciferase and 1 µg of GAL4-p300
deletion mutants in the presence or absence of MEKK1
. Results
represent the fold activation of the luciferase activity in the
presence of MEKK1
. Luciferase activity in the absence of MEKK1
was normalized to one. Results are representative of four experiments
and represent the mean ± S.D.
. As shown in Fig. 3, the
response of the large N-terminal region of p300 to MEKK1
was
mediated by two subdomains, aa 2-337 (N1, 32-fold) and aa 302-667
(N2, 89-fold) The N2 subdomain contains the C/H1 domain (aa 347-411)
and is three times more responsive than N1. Further deletions that
remove aa 302-406 (inclusive of the C/H1 domain) and aa 567-667 from
N2 did not significantly reduce its response to MEKK1, suggesting that
p300 aa 407-566 (N3) is sufficient to mediate part of the response of
p300 to MEKK1
. Taken together, these results suggest that there are
at least two transactivation domains within the N terminus that are
responsive to MEKK1
, p300 aa 2-337 and p300 aa 407-566, and that
the C/H1 domain is not involved in the N-terminal response of p300 to
MEKK1
.
enhanced the transcriptional activity of GAL4-p300 (aa 1737-2414) (C1) 32-fold. In contrast, MEKK1
only weakly activated the transcriptional activity of
GAL4-p300 (aa 1945-2414) (C2). These results suggest that p300 aa
1737-1945 is an important domain within the C-terminal half of p300
for responding to MEKK1
. Amino acids 1737-1945 of p300 contain the carboxyl portion of the C/H3 domain (aa 1653-1817) that is rich in
cysteines and histidines (12). The C/H3 domain has been shown to be an
important surface that mediates a number of important protein-protein
interactions (12, 21, 41-43). The above data suggest that the
C-terminal domain of p300 that mediates the response to MEKK1 may
reside somewhere between aa 1737 and 1945. We therefore tested a
GAL4-p300 construct that includes this region (GAL4-p300 aa 1709-1913)
(C3). As shown in Fig. 3, MEKK1
activates transcription mediated by
GAL4-p300 C3 (aa 1709-1913) by 20-fold. This is a significant
transcriptional induction, although slightly less than the fold of
activation (32-fold) observed for GAL4-p300 C1 (aa 1737- 2414) in
response to MEKK1
. Thus, a region including part of the C/H3 domain
of p300 is involved in the transcriptional response of the C-terminal
region of p300 to MEKK1
.
-mediated activation of GAL4-p300-mediated
transcription. As shown in Fig. 4, JNK1
(APF) was unable to block MEKK1
induction of transcription by either GAL4-p300N1 (aa 2-337), GAL4-p300N2 (aa 302-667), or GAL4-p300C1 (aa
1737-2414). In contrast, the dominant negative JNK1 mutant APF
potently inhibited GAL4-cJun-mediated transcription 8-fold as expected
(Fig. 4). These finding therefore suggest that JNK is not
necessary for MEKK1 to enhance p300-mediated transcription. MEKK1 has
also been shown to activate the I
B
kinases (34). By using a
dominant negative form of the I
B
kinase (S32A/S36A), we found no
evidence that this kinase is involved in mediating the ability of
MEKK1
to enhance p300-mediated transcription (data not shown).
View larger version (15K):
[in a new window]
Fig. 4.
Effect of dominant negative JNK on the
ability of MEKK1 to induce GAL4-p300-mediated
transcription. HeLa cells were transfected with 1 µg of MEKK1
and GAL4-luciferase along with either GAL4-p300 aa 2-337, GAL4-p300 aa
302-667, GAL4-p300 aa 1737-2414, or GAL4-cJun 1-223 (1 µg each) in
the presence or absence of JNK APF. Results are representative of three
experiments, and the mean ± S.D. is indicated.
View larger version (48K):
[in a new window]
Fig. 5.
Phosphorylation of GST-p300 domains in
vitro by recombinant JNK. GST-p300 aa 2-337, GST-p300
aa 302-667, and GST-p300 aa 1709-1913 were purified as described
under "Experimental Procedures" and standardized according to
protein concentration. Recombinant JNK was then used to phosphorylate
GST-p300 fragments in the presence of [ -32P]ATP at
37 °C. Reactions were terminated after 30 min by adding sample
loading buffer and running on SDS-PAGE. The gel was then dried and
autoradiographed. * indicates the positions of the GST or GST-p300
fusion proteins.
View larger version (15K):
[in a new window]
Fig. 6.
A, description of GST-p300 1709-1913
alanine mutants. Proline-directed serines or threonines of GST-p300 aa
1709-1913 were mutated to alanines in clusters (designated
A-D) as described under "Experimental Procedures." The
position of the serines (S) or threonines (T)
mutated are indicated by the asterisks. Numbers
represent the amino acid residue. B, phosphorylation of
wild-type and mutant p300. Phosphorylation of wild-type (WT)
GST-p300 aa 1709-1913 (lane 1), GST-p300 aa 1709-1913
Ala-1726/1849/1851/1854/1857 (Mut A, lane 2),
GST-p300 aa 1709-1913 Ala-1865/1868 (Mut B, lane
3), or GST-p300 aa 1709-1913
Ala-1726/1849/1851/1854/1857/1865/1868 (Mut A/B, lane
4) by recombinant JNK in vitro. GST-p300 mutants were
purified as described under "Experimental Procedures" and
phosphorylated by recombinant JNK in the presence of
[ -32P]ATP at 37 °C. Equal amounts of GST-p300
fusion proteins were used as substrates. Reactions were terminated by
adding sample loading buffer and running on SDS-PAGE. The gel was then
dried and autoradiographed. C, Coomassie Blue-stained gel of
GST-p300 aa 1709-1913 wild-type or alanine mutants. D,
activation of GAL4-p300 aa 302-667 and GAL4-p300 aa 1709-1913 ala
mutants by MEKK1
. HeLa cells were transfected with GAL4-luciferase,
wild-type GAL4-p300 aa 302-667, wild-type GAL4-p300 aa 1709-1913,
GAL4-p300 aa 302-667 Ala-317/499/512/524/594, or GAL4-p300 aa
1709-1913 Ala-1726/1849/1851/1854/1857/1865/1868/1878/1906/1909 (1 µg each) in the presence or absence of MEKK1
. Results are
representative of three experiments, and the mean ± S.D. is
indicated. Luciferase activity in the absence of MEKK1
was
normalized to one.
compared with its wild-type counterpart. Although the N- terminal subdomain of p300, N2 (aa 302-667), is not
phosphorylated by JNK1 in vitro (Fig. 5), we nevertheless
mutated all possible JNK sites in this domain and analyzed the effect
the mutations. As expected, mutation of the potential JNK sites in the
N2 subdomain had little effect on its ability to mediate MEKK1-induced
transcriptional activation (Fig. 6D). Taken together, these
results support the notion that the downstream kinase of MEKK1,
JNK1, is not essential for MEKK1-stimulated transcriptional activation
mediated by p300.
B
, we considered the possibility that MEKK1
may be able to
directly phosphorylate p300. Baculovirus MEKK1
was purified as
described previously (34) and used in kinase assays with GST-p300
fragments as substrates. Fig.
7A shows that baculovirus
MEKK1
strongly phosphorylated GST-p300 aa 302-667 (lane
3) as well as GST-p300 aa 300-528 (data not shown). As a control,
MEKK1
did not phosphorylate GST alone as shown in lane 1 (Fig. 7A). The ability of MEKK1 to phosphorylate GST-p300 aa 302-667 was also observed in immunocomplex kinase assays with MEKK1
(data not shown). In contrast, GST-p300 aa 2-337 (N1) (lane 2) and GST-p300 aa 1709-1913 (C3) (lane 4) were weakly
phosphorylated by MEKK1
(compare lanes 2 and 4 with lane 3). The phosphorylation of GST-p300 aa 302-667 as
opposed to GST-p300 aa 2-337 and GST-p300 aa 1709-1913 was not due to
differences in protein amount as Fig. 7B shows relatively
equal levels of protein by Coomassie Blue staining. These results are
consistent with the possibility that stimulation of GAL4-p300 aa
302-667-mediated transcription by MEKK1
may occur via direct
phosphorylation of p300 aa 302-667 by MEKK1. Furthermore, the
inability of MEKK1 to phosphorylate GST-p300 N1 and GST-p300 C3 (Fig.
7A, lanes 2 and 4) suggests that a yet
unidentified MEKK1-directed kinase other than JNK may be involved in
activating GAL4-p300-mediated transcription.
View larger version (54K):
[in a new window]
Fig. 7.
A, in vitro phosphorylation of p300
domains by purified MEKK1 . GST, GST-p300 aa 2-337, GST-p300 aa
302-667, and GST-p300 aa 1709-1913 were purified as described under
"Experimental Procedures" and standardized according to protein
concentration. Purified baculovirus MEKK1
(0.5 µg) was then used
to phosphorylate GST-p300 fragments in the presence of
[
-32P]ATP at 37 °C. Reactions were terminated by
adding sample loading buffer and running on SDS-PAGE. The gel was then
dried and autoradiographed. * indicates the positions of the GST or
GST-p300 fusion proteins. Autophosphorylated MEKK1
is indicated by
the arrow. B, Coomassie Blue-stained gel of GST
or GST-p300 fusion proteins. * indicates positions of the GST or
GST-p300 fusion proteins. Lower bands represent breakdown
products of the GST-p300 fusion proteins.
present almost exclusively in the nucleus in a punctate
pattern similar to that of the endogenous p300 (compare Fig.
8, E with F). In
fact, upon close inspection of the individual and merge images, the
active MEKK1 signal appears to overlap with that of p300, suggesting
that these two proteins my colocalize in the nucleus. This finding is
consistent with a direct regulatory role of MEKK1
on p300 in the
nucleus.
View larger version (40K):
[in a new window]
Fig. 8.
Analyses of the subcellular
localization of MEKK1 and p300. U2OS cells were transfected with
10 µg of HA-MEKK1 (A-D) or FLAG-MEKK1
(E-H) and double-stained with either anti-HA monoclonal
plus anti-p300 polyclonal (A and B) or anti-FLAG
monoclonal plus anti-p300 polyclonal (E and F)
antibodies as indicated. The positive transfectants (white
arrowhead) and untransfected neighboring cells (open
arrows) are indicated. A, untransfected (open
arrow) and neighboring cells transfected with HA-MEKK1
(arrowhead) were stained with an HA epitope antibody.
B, the same cells as in A stained with anti-p300
polyclonal antibodies. C, merged image of A and
B in which an arrow indicates a cell in which
p300 is found in the cytoplasm with a punctate pattern identical to
that of MEKK1. E, untransfected (open arrow) and
a neighboring cell transfected with FLAG-MEKK1
(arrowhead) were stained with a anti-FLAG epitope antibody.
F, the same cells as in E stained with p300
polyclonal antibodies. G, merged image of E and
F. D and H are DAPI stainings that
identify the nuclei of the cells.
(36% apoptotic cells
for inactive ribozyme MCF-7 versus 16% apoptotic cells for
p300 active ribozyme MCF-7). This suggests that p300 may be part of the
pathway by which MEKK1 induces apoptosis and suggests that the
modification of the transcriptional activity of p300 by MEKK1 may be
important for this process.
View larger version (18K):
[in a new window]
Fig. 9.
Effect of p300 ribozyme on the ability of
MEKK1 to induce apoptosis in MCF-7 cells.
10 µg of pCDNA3 vector, pCDNA3 MEKK1
, or pCDNA3
MEKK1
K432M were cotransfected along with GFP into MCF-7 cells
containing an inactive or active p300 ribozyme. Forty eight hours after
transfection, 400 cells were counted, and the percentage of apoptotic
cells were scored as described under "Experimental Procedures."
Results are representative of two experiments, and the mean ± S.D. of triplicate cultures is indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (47) and MEKK1 activates I
B
kinase
which activates NF
B. We thus considered whether MEKK1 merely
activated this pathway that facilitated the interaction of NF
B with
GAL4-p300 and somehow resulted in transcriptional activation. We tested
this possibility using the dominant negative form of the I
B
kinase (34) and found no evidence for the involvement of NF
B in the
stimulation of p300-mediated transcription by MEKK1 (data not shown).
) in the nucleus of transfected cells and
its localization appears to coincide with that of the endogenous p300 (Fig. 8, E and F). Furthermore, endogenous p300
can be found colocalizing with the punctate cytoplasmic MEKK1 signals
in cells overexpressing the full-length inactive form of MEKK1, which
resides in the cytoplasm, suggesting a possible physical interaction.
Collectively, these findings are consistent with the possibility of
direct regulation of p300 by MEKK1.
, suggesting that p300 may be an important
component by which MEKK1
induces apoptosis. Our findings thus
identify MEKK1 as a kinase that may regulate the transcriptional as
well as the biological activities of p300.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Michael Greenberg and Azad Bonni for critical reading of the manuscript and Melanie Cobb for helpful discussion and suggestions. We thank Hans Peter Hefti and MyungSoo Joo for making GAL4-p300 aa 407-566 and GAL4-p300 aa 302-667, respectively. We thank Kenneth Liu for computer assistance. We also thank many colleagues for providing reagents used in this study.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant GM58012 (to Y. 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.
Supported by a Spanish Government fellowship and by a Taplin
Postdoctoral fellowship.
¶ To whom correspondence should be addressed: Dept. of Pathology, Harvard Medical School, Warren Alpert Bldg., Rm. 120, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-432-4318; Fax: 617-432-1313; E-mail: yang_shi@hms.harvard.edu.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M008113200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CBP, CREB-binding protein; CREB, cAMP-responsive element-binding protein; JNK, c-Jun N- terminal kinase; MEKK1, mitogen-activated/extracellular response kinase kinase; aa, amino acid; GST, glutathione S-transferase; C/H, cysteine/histidine; HAT, histone acetyltransferase; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; HA, hemagglutinin; DAPI, 4,6-diamidino-2-phenylindole; MOPS, 3-(N-morpholino)propanesulfonic acid; aa, amino acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Harlow, E., Whyte, P., Franza, B. R., Jr., and Schley, C. (1986) Mol. Cell. Biol. 6, 1579-1589[Medline] [Order article via Infotrieve] |
2. | Yee, S. P., and Branton, P. E. (1985) Virology 147, 142-153[Medline] [Order article via Infotrieve] |
3. | Arany, Z., Newsome, D., Oldread, E., Livingston, D. M., and Eckner, R. (1995) Nature 374, 81-84[CrossRef][Medline] [Order article via Infotrieve] |
4. | Kwok, R. P. S., Lundblad, J. R., Chrivia, J. C., Richards, J. P., Bachinger, H. P., Brennan, R. G., Roberts, S. G. E., Green, M. R., and Goodman, R. H. (1994) Nature 370, 223-226[CrossRef][Medline] [Order article via Infotrieve] |
5. | 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] |
6. | Egan, C., Bayley, S. T., and Branton, P. E. (1989) Oncogene 4, 383-388[Medline] [Order article via Infotrieve] |
7. | Wang, H.-G. H., Rikatake, Y., Carter, M. C., Yaciuk, P., Abraham, S. E., Brad, Z., and Moran, E. (1993) J. Virol. 67, 476-488[Abstract] |
8. | Whyte, P., Williamson, N. M., and Harlow, E. (1989) Cell 56, 67-75[Medline] [Order article via Infotrieve] |
9. | Jelsma, T. N., Howe, J. A., Mymryk, J. S., Evelegh, C. M., Cunnif, N. F., and Bayley, S. T. (1989) Virology 171, 120-130[Medline] [Order article via Infotrieve] |
10. | Moran, E. (1993) Curr. Opin. Genet. & Dev. 3, 63-70[Medline] [Order article via Infotrieve] |
11. | Mymryk, J. S., Lee, R. W. H., and Bayley, S. T. (1992) Mol. Biol. Cell 3, 1107-1115[Abstract] |
12. | Eckner, R., Ewen, M. E., Newsome, D., Gerdes, M., DeCaprio, J. A., Lawrence, J. B., and D. M., L. (1994) Genes Dev. 8, 869-884[Abstract] |
13. |
Shi, Y.,
and Mello, C.
(1998)
Genes Dev.
12,
943-955 |
14. | Akimaru, H., Chen, Y., Dai, P., Hou, D.-X., Nonaka, M., Smolik, S. M., Armstrong, S., Goodman, R. H., and Shunsuke, I. (1997) Nature 386, 735-738[CrossRef][Medline] [Order article via Infotrieve] |
15. | Yao, T.-P., Oh, S. P., Fuchs, M., Zhou, N.-D., Ch'ng, L.-E., Newsome, D., Bronson, R. T., Li, E., Livingston, D. M., and Eckner, R. (1998) Cell 93, 361-372[Medline] [Order article via Infotrieve] |
16. | Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H., and Nakatani, Y. (1996) Cell 87, 953-959[Medline] [Order article via Infotrieve] |
17. | Bannister, A. J., and Kouzarides, T. (1996) Nature 384, 641-643[CrossRef][Medline] [Order article via Infotrieve] |
18. | Gu, W., and Roeder, R. G. (1997) Cell 90, 595-606[Medline] [Order article via Infotrieve] |
19. |
Liu, L.,
Scolnick, D. M.,
Trievel, R. C.,
Zhang, H. B.,
Marmorstein, R.,
Halazonetis, T. D.,
and Berger, S. L.
(1999)
Mol. Cell. Biol.
19,
1202-1209 |
20. | Imhof, A., Yang, X.-J., Ogryzko, V. V., Nakatani, Y., Wolffe, A. P., and Ge, H. (1997) Curr. Biol. 7, 689-692[Medline] [Order article via Infotrieve] |
21. |
Yuan, W.,
Condorelli, G.,
Caruso, M.,
Felsani, A.,
and Giordano, A.
(1996)
J. Biol. Chem.
271,
9009-9013 |
22. |
Lee, J.-S.,
Zhang, X.,
and Shi, Y.
(1996)
J. Biol. Chem.
271,
17666-17674 |
23. | Nakajima, T., Uchida, C., Anderson, S. F., Lee, C.-G., Hurwitz, J., Parvin, J. D., and Montminy, M. (1997) Cell 90, 1107-1112[Medline] [Order article via Infotrieve] |
24. | Kitabayashi, I., Eckner, R., Arany, Z., Chiu, R., Gachelin, G., Livingston, D. M., and Yokoyama, K. K. (1995) EMBO J. 14, 3496-3509[Abstract] |
25. | Janknecht, R., and Nordheim, A. (1996) Biochem. Biophys. Res. Commun. 228, 831-837[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Swope, D. L.,
Mueller, C. L.,
and Chrivia, J. C.
(1996)
J. Biol. Chem.
271,
28138-28145 |
27. | Barbeau, D., Charbonneau, R., Whalen, S. G., Bayley, S. T., and Branton, P. E. (1994) Oncogene 9, 359-373[Medline] [Order article via Infotrieve] |
28. | Cardone, M. H., Salvesen, G. S., Widmann, C., Johnson, G., and Frisch, S. M. (1997) Cell 90, 315-323[Medline] [Order article via Infotrieve] |
29. |
Widmann, C.,
Gerwins, P.,
Johnson, N. L.,
Jarpe, M. B.,
and Johnson, G. L.
(1998)
Mol. Cell. Biol.
18,
2416-2429 |
30. | Sanchez, I., Hughes, R. T., Mayer, B. J., Yee, K., Woodgett, J. R., Avruch, J., Kyriakis, J. M., and Zon, L. I. (1994) Nature 372, 794-798[Medline] [Order article via Infotrieve] |
31. | Yan, M., Dai, T., Deak, J. C., Kyriakis, J. M., Zon, L. I., Woodgett, J. R., and Templeton, D. J. (1994) Nature 372, 798-800[Medline] [Order article via Infotrieve] |
32. | Derijard, B., Hibi, M., Wu, I.-H., Barrett, T., Su, B., Deng, T., Karin, M., and Davis, R. J. (1994) Cell 76, 1-20[Medline] [Order article via Infotrieve] |
33. | Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E. A., Ahmad, M. F., Avruch, J., and Woodgett, J. R. (1994) Nature 369, 156-160[CrossRef][Medline] [Order article via Infotrieve] |
34. | Lee, F. S., Hagler, J., Chen, Z. J., and Maniatis, T. (1997) Cell 88, 213-222[Medline] [Order article via Infotrieve] |
35. |
Yujiri, T.,
Sather, S.,
Fanger, G. R.,
and Johnson, G. L.
(1998)
Science
282,
1911-1914 |
36. |
Xia, Y.,
Makris, C.,
Su, B.,
Li, E.,
Yang, J.,
Nemerow, G. R.,
and Karin, M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5243-5248 |
37. | Lee, J.-S., Galvin, K. M., See, R. H., Eckner, R., Livingston, D., Moran, E., and Shi, Y. (1995) Genes Dev. 9, 1188-1198[Abstract] |
38. |
Ronco, L. V.,
Karpova, A. Y.,
Vidal, M.,
and Howley, P. M.
(1998)
Genes Dev.
12,
2061-2072 |
39. |
See, R. H.,
and Shi, Y.
(1998)
Mol. Cell. Biol.
18,
4012-4022 |
40. |
Yuan, Z.-M.,
Huang, Y.,
Ishiko, T.,
Nakada, S.,
Utsugisawa, T.,
Shioya, H.,
Utsugisawa, Y.,
Yokoyama, K.,
Weichselbaum, R.,
Shi, Y.,
and Kufe, D.
(1999)
J. Biol. Chem.
274,
1883-1886 |
41. | Avanaggiati, M. L., Ogryzko, V., Gardner, K., Giordano, A., Levine, A. S., and Kelly, K. (1997) Cell 89, 1175-1184[Medline] [Order article via Infotrieve] |
42. | Eckner, R., Ludlow, J. W., Lill, N. L., Oldread, E., Arany, Z., Modjtahedi, N., DeCaprio, J. A., Livingston, D. M., and Morgan, J. A. (1996) Mol. Cell. Biol. 16, 3454-3464[Abstract] |
43. |
Kurokawa, R.,
Kalafus, D.,
Ogliastro, M.-H.,
Kioussi, C.,
Xu, L.,
Torchia, J.,
Rosenfeld, M. G.,
and Glass, C. K.
(1998)
Science
279,
700-703 |
44. | Derijard, B., Raingeaud, J., Barrett, I.-H., Han, J., Ulevitch, R. J., and Davis, R. J. (1995) Science 267, 682-685[Medline] [Order article via Infotrieve] |
45. | Widmann, C., Johnson, N. L., Gardner, A. M., Smith, R. J., and Johnson, G. L. (1997) Oncogene 15, 2439-2447[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Johnson, N. L.,
Gardner, A. M.,
Diener, K. M.,
Lange-Carter, C. A.,
Gleavy, J.,
Jarpe, M. B.,
Minden, A.,
Karin, M.,
Zon, L. I.,
and Johnson, G. L.
(1996)
J. Biol. Chem.
271,
3229-3237 |
47. |
Gerritsen, M. E.,
Williams, A. J.,
Neish, A. S.,
Moore, S.,
Shi, Y.,
and Collins, T.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2927-2932 |
48. |
Yan, M.,
and Templeton, D. J.
(1994)
J. Biol. Chem.
269,
19067-19073 |
49. |
Blank, J. L.,
Gerwins, P.,
Elliott, E. M.,
Sather, S.,
and Johnson, G. L.
(1996)
J. Biol. Chem.
271,
5361-5368 |
50. |
Fuchs, S. Y.,
Adler, V.,
Pincus, M. R.,
and Ronai, Z.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
10541-10546 |
51. | Lill, N. L., Grossman, S. R., Ginsberg, D., DeCaprio, J., and Livingston, D. M. (1997) Nature 387, 823-827[CrossRef][Medline] [Order article via Infotrieve] |
52. | Gu, W., Shi, X.-L., and Roeder, R. G. (1997) Nature 387, 819-823[CrossRef][Medline] [Order article via Infotrieve] |