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Molecular and Cellular Biology, September 2000, p. 6612-6625, Vol. 20, No. 17
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The SMRT Corepressor Is Regulated by a MEK-1 Kinase Pathway:
Inhibition of Corepressor Function Is Associated with SMRT
Phosphorylation and Nuclear Export
Suk-Hyun
Hong and
Martin L.
Privalsky*
Section of Microbiology, University of
California at Davis, Davis, California 95616
Received 13 December 1999/Returned for modification 29 February
2000/Accepted 24 May 2000
 |
ABSTRACT |
The SMRT (silencing mediator of retinoic acid and thyroid hormone
receptor) corepressor participates in the repression of target gene
expression by a variety of transcription factors, including the nuclear
hormone receptors, promyelocytic leukemia zinc finger protein, and
B-cell leukemia protein 6. The ability of SMRT to associate with these
transcription factors and thereby to mediate repression is strongly
inhibited by activation of tyrosine kinase signaling pathways, such as
that represented by the epidermal growth factor receptor. We report
here that SMRT function is potently inhibited by a mitogen-activated
protein kinase (MAPK) kinase kinase (MAPKKK) cascade that operates
downstream of this growth factor receptor. Intriguingly, the SMRT
protein is a substrate for phosphorylation by protein kinases operating
at multiple levels in this MAPKKK pathway, including the MAPKs,
MAPK-extracellular signal-regulated kinase 1 (MEK-1), and MEK-1 kinase
(MEKK-1). Phosphorylation of SMRT by MEKK-1 and, to a lesser extent,
MEK-1 inhibits the ability of SMRT to physically tether to its
transcription factor partners. Notably, activation of MEKK-1 or MEK-1
signaling in transfected cells also leads to a redistribution of the
SMRT protein from a nuclear compartment to a more perinuclear or
cytoplasmic compartment. We suggest that SMRT-mediated repression is
regulated by the MAPKKK cascade and that changes both in the affinity
of SMRT for its transcription factors and in the subcellular
distribution of SMRT contribute to the loss of SMRT function that is
observed in response to kinase signal transduction.
 |
INTRODUCTION |
Eukaryotic transcription factors can
exert both positive and negative effects on gene expression. A number
of transcriptional regulators are, in fact, bipolar in their
properties, with a given transcription factor being able to both
repress and activate target gene expression. Perhaps the most
extensively analyzed of these bipolar transcription factors are the
nuclear hormone receptors, such as the retinoic acid receptors (RARs)
and the thyroid hormone receptors (T3Rs) (2, 31, 37, 43,
64). RARs and T3Rs are ligand-regulated transcription factors
that bind to specific target promoter sequences, denoted hormone
response elements, in both the absence and the presence of cognate
hormone. In the absence of hormone, these nuclear receptors typically
repress gene transcription; conversely, binding of cognate hormone
converts the nuclear receptors into strong transcriptional activators
(2, 31, 37, 43, 64).
RARs and T3Rs manifest these divergent transcriptional properties
through their ability to recruit auxiliary polypeptides, denoted
corepressors and coactivators (12, 22, 36, 62, 76). In the
absence of hormone ligand, RARs and T3Rs are able to bind to two
interrelated corepressor polypeptides, denoted SMRT (silencing mediator
of retinoic acid and thyroid hormone receptor) and N-CoR (nuclear
hormone receptor corepressor) (11, 21, 29, 35, 45, 46, 51, 55, 73,
79, 80); SMRT and N-CoR recruit, in turn, a larger corepressor
complex including mSin3, RbAp-46, RbAp-48, SAP-18, and SAP-30, and
histone deacetylases (5, 17, 47, 71). Conversely, binding of
hormone ligand results in a conformational change in the nuclear
hormone receptors that leads to release of the corepressor complex and
recruitment of a series of coactivator complexes, many of which possess
histone acetyltransferase activity (12, 22, 36, 62, 74, 76). The precise mechanisms by which corepressors and coactivators modulate
transcription remain to be fully elucidated but appear to involve both
modifications of the chromatin template and interactions with the
general transcriptional machinery (5, 17, 28, 36, 41, 58, 59, 62,
72, 74-76).
Despite the important regulatory role of hormone ligand in T3R and RAR
function, these nuclear receptors actually function as a molecular
nexus at which a variety of both hormonal and nonhormonal signals
converge to generate combinatorial regulation of target gene
expression. Therefore, the ultimate transcriptional response mediated
by nuclear hormone receptors is determined not just by the hormone
status but also by the nature of the target promoter and by the actions
of nonligand signal transduction pathways operative in the cell
(26, 39, 43, 44, 56, 69). Particularly of note is the
ability of certain protein kinases to modulate, both negatively and
positively, nuclear hormone receptor function (reviewed in references
7, 8, 26, 56, and 69). The actions of these kinases can, for example, induce target gene expression by nuclear hormone receptors even in the absence of ligand
or can further enhance the activation observed in the presence of
hormone ligand. In some cases, these effects appear to be mediated through direct phosphorylation of the receptor itself (3, 10, 16,
23, 24, 27, 33, 63, 65). In other contexts, however, the
mechanism by which these protein kinases alter the regulatory
properties of the nuclear hormone receptor is not known but does not
appear to involve modification of the receptor itself (6, 20, 52,
67, 69).
Might auxiliary factors, such as corepressors, serve as regulatory
targets for these protein kinase signal transducers? We and others have
reported that the SMRT and N-CoR corepressors are important targets of
regulation by the epidermal growth factor (EGF) receptor and by cyclic
AMP (20, 30, 67). EGF receptor signaling, for example, has
little or no effect on T3R-mediated activation but strongly counteracts
T3R-mediated repression, apparently by interfering with the
ability of the SMRT or N-CoR corepressor to interact with the
nuclear hormone receptor (20, 30). EGF receptor signaling
similarly interferes with corepressor recruitment and transcriptional
repression by RAR and by PLZF (promyelocytic leukemia zinc finger
protein), a nonreceptor transcriptional repressor that also utilizes
the SMRT-N-CoR corepressor complex (20). The
signaling events and mechanisms by which the EGF receptor regulates corepressor function were not previously determined. Here, we
report that SMRT corepressor function is regulated by components
of a mitogen-activated protein kinase (MAPK) kinase kinase
(MAPKKK) cascade that operates downstream of the EGF receptor. SMRT
appears to be a substrate for phosphorylation by multiple components of
this kinase cascade, including MAPK-extracellular signal-regulated
kinase (ERK) kinase 1 (MEK-1), MEK-1 kinase (MEKK-1), p38, and possibly
additional downstream protein kinases. SMRT phosphorylation in response
to the actions of MEKK-1 or, to a somewhat lesser extent, MEK-1
strongly inhibits the ability of the corepressor to mediate repression
by nuclear hormone receptors and by other transcription factors, such
as PLZF. This MEKK-1 or MEK-1 phosphorylation of SMRT is closely
paralleled by an inhibition of the ability of the corepressor to bind
to nuclear receptors and by a relocalization of the SMRT corepressor
from the nucleus to the cytoplasm of the cell. Our observations suggest
that the MAPKKK pathway serves as a potent negative regulator of the
SMRT corepressor and that this regulation appears to operate, at least in part, by altering both the subcellular location and the receptor interaction properties of the corepressor protein.
 |
MATERIALS AND METHODS |
Plasmid constructs.
The pCMV-SMRT and pCMV-SMRT-C vectors
were constructed by inserting EcoRI fragments from the
previously described pSG5-SMRT TRAC-2 and TRAC-1 constructs
(51) into a pCR3.1 vector (Invitrogen, Carlsbad, Calif.).
Mammalian two-hybrid vectors for various SMRT and T3R
derivatives
were constructed as previously described (18, 20). The
pSG5-GAL4 activation domain (AD) (pSG5-GAL4AD)-RAR
and retinoid X
receptor
(RXR
) vectors were constructed by inserting EcoRV and XhoI fragments, generated by PCR, into
the pSG5-GAL4AD vector. Construction of the glutathione
S-transferase (GST) and GST-T3R
vectors was described
previously (72). The green fluorescent protein (GFP)-SMRT
vector was constructed by inserting the
BsrGI-EcoRI (blunt) fragment from pSG5-SMRT into
the BsrGI-HindIII (blunt) sites on a
CMVs-GFPTYGBH vector. Base substitution mutations, designed to abolish
MAPK sites within the SMRT sequence, were created by standard
PCR-mediated in vitro mutagenesis methodologies.
Expression vectors for full-length MEKK-1 and MEK-1 and for dominant
negative MEK-1 expression plasmids were obtained from Chris Jamieson
(University of California at San Francisco). The pMT3-HA-SAPK (p46
),
pMT3-HA-p38, pEBG-SEK1, pMT3-ERK-1, pCMV5-FLAG-MEKK-1(817-1493), and
pCMV5-FLAG-MEKK-1-KM(817-1493) clones were obtained from John Kyriakis
(Massachusetts General Hospital). Baculovirus stocks of recombinant
His6-MEKK-1
were obtained from Tom Maniatis (Harvard University).
Transient transfections.
CV-1 cell transfections were
performed by a Lipofectin-mediated method using the general protocol
recommended by the manufacturer (GIBCO/BRL Life Technologies,
Rockville, Md.). Approximately 4 × 105 cells were
transfected with 50 ng of pSG5-T3R
or pSG5-v-ErbA plasmid, 100 ng of
pCMV-lacZ or pCH110 as an internal control, and 100 ng of a ptk-luc-TRE
reporter, together with expression vectors for the various signal
transducers tested here (equal quantities of the equivalent empty
vectors were substituted as appropriate). The cells were transferred to
serum-free Dulbecco modified Eagle medium 24 h after transfection
and harvested 24 h later. The luciferase and
-galactosidase
assays were performed as previously described, and the relative
luciferase activity was determined (19, 20).
Mammalian two-hybrid assays.
Exponentially growing CV-1
cells (7 × 104 cells per well in 12-well culture
plates) were transiently transfected by Lipofectin methodology with 25 ng of the appropriate pSG5-GAL4 DNA binding domain (DBD) (pSG5-GAL4DBD)
vector, 100 ng of the appropriate pSG5-GAL4AD vector, 100 ng of the
pGL2-GAL4-17-mer luciferase reporter, 100 ng of the pCMV-lacZ internal
control, and appropriate expression vectors for the various signal
transducers tested here (or equal quantities of the equivalent empty
vectors as appropriate) (20). The cells were transferred to
serum-free Dulbecco modified Eagle medium 24 h after transfection
and harvested an additional 24 h later. The luciferase and
-galactosidase assays were performed as previously described, and
the relative luciferase activity was determined (20).
Immunoblotting.
CV-1 cells (7 × 104 per
well) were transfected with the appropriate expression vectors,
harvested 48 h after transfection by scraping, and lysed by being
mixed with sodium dodecyl sulfate (SDS) electrophoresis sample buffer.
The lysates were then sonicated to reduce viscosity, boiled for 5 min,
and loaded onto an SDS-7.5% polyacrylamide-0.3% bisacrylamide gel.
After electrophoresis, the proteins were electrophoretically
transferred to a nitrocellulose membrane. The membrane was incubated in
blocking buffer (5% nonfat dry milk in TBST [0.1% Tween 20, 150 mM
NaCl, 10 mM Tris-Cl, pH 7.6]) for 1 h, and then incubated with
appropriate primary antibodies (diluted in 5% bovine serum albumin in
TBST) for 1 h. The membrane was next washed extensively with
TBST and incubated with appropriate secondary antibodies
(horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse
antibodies [Affinity BioReagents, Golden, Colo.]; diluted
1:2,000). After extensive washing with TBST, the chemiluminescent Western detection system was used for visualization of the
immunoreactive proteins as specified by the manufacturer (New England
Biolabs, Beverly, Mass.).
Phosphorylation-dephosphorylation assays.
CV-1 cells
(2.5 × 105) were transfected with the pCMV-SMRT-C,
MEKK-1, MEK-1, MEKK-1 kinase mutant (MEKK1KM), or dominant negative MEK-1 expression vectors. The cells were harvested 48 h later by
scraping and centrifugation in 150 µl of whole-cell extraction buffer
(25 mM HEPES [pH 7.5], 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1% Triton X-100, 0.1 mM dithiothreitol [DTT], 0.5 mM
phenylmethylsulfonyl fluoride, complete protease inhibitor cocktail
[Boehringer GmbH, Mannheim, Germany]). Lysates were then incubated in
the presence or absence of 0.5 U of calf intestinal alkaline
phosphatase (New England Biolabs) for 30 min at 30°C. The incubation
reactions were terminated by mixing the samples with SDS sample buffer. The samples were boiled for 5 min, loaded onto an SDS-7.5%
polyacrylamide-0.3% bisacrylamide gel, and subjected to
electrophoresis and immunoblotting as described above.
Coimmunoprecipitation assays.
Approximately 7.5 × 105 COS-1 cells were transfected with various
combinations of pCMV-SMRT, pCMV-FLAG-MEKK-1, or pCMV-HA-MEK-1 expression vectors by use of a Lipofectamine-Plus procedure (GIBCO/BRL Life Technologies). Whole-cell lysates were prepared by gently sonicating the cells in 600 µl of immunoprecipitation buffer (IP buffer; phosphate-buffered saline, 1 mM EDTA, 1.5 mg of iodoacetamide per ml, 100 µM Na3VO4, 0.5% Triton X-100, 20 mM
-glycerolphosphate, 0.2 mM phenylmethylsulfonyl fluoride,
complete protease inhibitor cocktail). After clarification by 5 min of
centrifugation in a microcentrifuge at 4°C, the resulting supernatant
was incubated for 1 h at 4°C with 2.4 µl of anti-FLAG antibody
M2 (Sigma Chemical Co., St. Louis, Mo.; diluted 1:250). Fifteen
microliters of protein A-Sepharose (as a 50% slurry in IP buffer;
Sigma) was then added, and the samples were incubated with continuous
mixing for an additional 1 h at 4°C. The protein A-Sepharose
matrix was extensively washed with IP buffer, and any proteins
remaining bound to it were eluted with SDS sample buffer and detected
by Western analysis.
In vitro kinase assays.
GST-SMRT fusion proteins were
expressed in Escherichia coli and immobilized on
glutathione-agarose as previously described (19, 20).
GST-SMRT proteins were then incubated with 0.4 µg of MEKK-1 (purified
from recombinant E. coli; Upstate Biotechnology, Lake
Placid, N.Y.), 0.5 U of activated MEK-1 (purified from recombinant E. coli; Upstate Biotechnology), or 0.5 µg of
His6-tagged
MEKK-1 (purified from baculovirus-infected
Sf9 cells) for 30 min at 30°C in 50 µl of kinase buffer (20 mM
HEPES [pH 7.5], 20 mM
-glycerolphosphate, 10 mM MgCl2,
10 mM p-nitrophenyl phosphate, 100 µM
Na3VO4, 2 mM DTT, 20 µM ATP) containing 5 µCi of [
-32P]ATP. GST-SEK-1 (stress-activated
protein kinase
[SAPK-
, also known as ERK kinase 1]) was used
in some experiments as a positive control. The kinase reactions were
terminated by adding SDS-sample buffer. The samples were boiled and
resolved by SDS-polyacrylamide gel electrophoresis (PAGE).
Phosphorylated proteins were visualized by autoradiography.
Immunocomplex kinase assays.
COS-1 cells were transfected
with a pCMV5-MEKK1 expression vector (0.7 µg) and lysed 48 h
later by gentle sonication in 600 µl of whole-cell extraction buffer.
The MEKK-1 protein was immunoprecipitated by the addition of 2.4 µl
of MEKK-1-directed antibodies. Immunopurified SMRT protein was
obtained from pCMV-SMRT-transfected COS-1 cells in a similar
manner with antibodies directed against SMRT (Affinity BioReagents). The immunopurified MEKK-1 preparation was then
tested for the ability to phosphorylate the immunopurified SMRT protein or GST-SMRT protein derivatives obtained from E. coli using
the in vitro kinase assay described above.
Receptor-corepressor binding assays in vitro.
GST-T3R
proteins were expressed in E. coli and purified and
immobilized by binding to a glutathione-agarose matrix (19, 20). 35S-radiolabeled SMRT-C (TRAC-1) proteins were
synthesized by a coupled in vitro transcription-translation system (TnT
kit; Promega, Madison, Wis.). The radiolabeled SMRT-C proteins were
incubated in kinase buffer (containing 20 µM unlabeled ATP) with
MEKK-1 or activated MEK-1 (each purified from recombinant E. coli) or without exogenous kinase. The SMRT-C proteins were then
incubated with the immobilized GST-T3R
polypeptides for 1 h at
4°C in 250 ml of HEMG buffer (51). The glutathione-agarose
matrix was extensively washed, and proteins remaining bound to the
matrix were eluted with free glutathione and analyzed by SDS-PAGE
(19, 20). The electrophoretograms were visualized by
autoradiography and quantified by PhosphorImager analysis (STORM;
Molecular Dynamics).
Laser scanning confocal microscopy.
Approximately
105 CV-1 cells were seeded in a chambered coverslip cell
culture system (Nalge-Nunc, Rochester, N.Y.). The cells were
transfected with the pCMV-GFP-SMRT vector together with an appropriate
expression vector for either v-ErbB, v-Ras, MEKK-1, or MEK-1 (or an
equivalent empty vector as a control) using the Lipofectamine-Plus
procedure. One day after transfection, the cells were transferred to
serum-free Dulbecco modified Eagle medium and incubated for an
additional 24 h. The subcellular location of the GFP-SMRT fusion
polypeptide was visualized using a Leica TCS-SP Ar/Kr/HeNe laser
scanning confocal microscope, with excitation at 488 nm and detection
at 500 to 540 nm.
Subcellular fractionation assays.
CV-1 cells (2.5 × 105) were transfected with pCMV-SMRT-C together with
expression vectors for the various signal transducers tested here using
the Lipofectamine-Plus protocol. After 48 h, the cells were
harvested in phosphate-buffered saline and incubated in hypotonic
buffer (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl,
0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT) for 10 min at 4°C.
Subsequently, the cells were lysed by 10 strokes of a Dounce
microhomogenizer with a loosely fitting plunger. The cell lysates were
then centrifuged at 2,000 × g for 5 min. The resulting
pellets (nuclear fraction) and supernatants (cytoplasmic fraction) were
subsequently solubilized in SDS sample buffer and analyzed by immunoblotting.
 |
RESULTS |
Tyrosine kinase signaling abrogates transcriptional repression and
interferes with the ability of SMRT to interact with T3R in vivo.
The effects of EGF receptor signaling on T3R-mediated gene regulation
were tested with a transient transfection assay using a luciferase
reporter containing a thyroid hormone response element (TRE). CV-1
cells possess few or no endogenous T3Rs and display a basal level of
luciferase reporter expression in either the absence or the presence of
triiodothyronine (T3) hormone (Fig. 1A).
As anticipated (11, 21, 51), the introduction of a T3R
expression vector into these cells in the absence of T3 hormone resulted in the repression of luciferase expression to below basal levels; the addition of T3 hormone reversed this repression and led to
T3R-mediated enhancement of luciferase expression to levels significantly above basal levels (Fig. 1A). Notably, cointroduction of
a constitutively active form of the EGF receptor, denoted v-ErbB, into
these cells reversed the repression mediated by T3R in the absence of
T3, with relatively little effect on the activation mediated by T3R in
the presence of T3 (Fig. 1A). The ability of EGF receptor signaling to
abrogate transcriptional repression extended to repression by PLZF, a
transcription factor that also utilizes the SMRT-N-CoR corepressor
complex but that is not a member of the nuclear receptor family
(20) (see Fig. 4C). Although v-ErbB was used in these
experiments as a means of uniformly activating EGF receptor signaling
in transfected cells (40, 66), analogous results were
observed upon induction of wild-type EGF receptor activity (data not
shown).

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FIG. 1.
Effect of EGF receptor-v-ErbB signaling on
T3R transcriptional repression and interactions with SMRT. (A)
Inhibition of T3R-mediated repression by v-ErbB. CV-1 cells were
transfected with empty plasmid pSG5 or plasmid pSG5-T3R in the
presence or absence of a v-ErbB expression plasmid, as indicated
below the panel. Cells were then incubated in the presence (+) or
absence ( ) of 1 µM T3, and the expression of a T3R-responsive (TRE)
luciferase reporter was determined relative to that of a constitutive
pCMV-lacZ reporter used as an internal control. (B) Interaction of
T3R with SMRT in a mammalian two-hybrid assay. A pSG5-GAL4DBD
plasmid containing no insert (empty-DBD), containing the RID
(amino acids 751 to 1495) of SMRT (DBD-SMRT), or containing SMRT
bearing a deletion of the RID (DBD-SMRT RID) was cotransfected into
CV-1 cells together with a GAL4-17-mer luciferase reporter and a
pSG5-GAL4AD construct. The pSG5-GAL4AD construct, indicated below the
panel, contained no insert (empty), the wild-type T3R ligand binding
domain (T3R), the T3R ligand binding domain with a P156R mutation
that disrupts SMRT association (P156), the analogous region of the
v-ErbA mutant form of T3R (T3R-vA), or the vitamin D3
receptor (VDR). The cells were incubated in the absence or presence of
1 µM cognate hormone, and luciferase activity was determined relative
to the -galactosidase activity of the pCH110 plasmid introduced as
an internal control. (C) v-ErbB inhibition of the two-hybrid
interaction between T3R and SMRT. The same mammalian two-hybrid
vectors as those described for panel B and GAL4DBD fused with the
ligand binding domain of RXR were cotransfected into CV-1 cells in
various combinations, as indicated for each panel. In addition,
the cells were cotransfected with the indicated amounts of the
v-ErbB expression plasmid (0 to 200 ng), the GAL4-17-mer luciferase
reporter, and a pCMV-lacZ internal control plasmid. The cells were
incubated in the absence of T3, and the relative luciferase activity
was determined. The average and standard deviation of duplicate
experiments are presented.
|
|
To examine if the abrogation of T3R- and PLZF-mediated
repression by EGF receptor-v-ErbB signaling reflected changes in SMRT
function, we next used a mammalian two-hybrid system as a measure
of
the ability of SMRT to interact with T3R in transfected cells
(
18,
20,
73). For this assay, a GAL4DBD-SMRT fusion construct,
a
GAL4AD-T3R

fusion construct, and a luciferase reporter bearing
GAL4
binding sites (GAL4-17-mer) were introduced separately or
in
combination into CV-1 cells. A strong activation of the GAL4-17-mer
luciferase reporter was observed when all three constructs were
introduced together, presumably reflecting the ability of the
SMRT and
T3R determinants to interact and thereby reconstitute
a functional GAL4
transcription factor (Fig.
1B). Supporting this
interpretation are the
following: (i) each of the fusion constructs
was transcriptionally
inactive when introduced separately; (ii)
T3 hormone abolished the T3R
interaction with SMRT both in vitro
and in the mammalian two-hybrid
system; (iii) mutants of T3R (P156R)
or other nuclear receptors, such
as vitamin the D
3 receptor, that
do not interact
significantly with SMRT in vitro (
51,
73)
did not interact
in the two-hybrid system; (iv) irrelevant proteins
or SMRT mutants
(

RID) that fail to interact with T3R in vitro
(
51) did
not interact with T3R in the two-hybrid system; and
(v) constitutive
repression mutants of T3R (T3R-vA) that bind
SMRT in a
hormone-independent fashion in vitro (
51) exhibited
a
hormone-independent interaction with SMRT in the two-hybrid
assay (Fig.
1B).
The ability of T3R to interact with SMRT in the mammalian two-hybrid
assay was severely compromised by cointroduction of the
EGF
receptor-v-ErbB construct (Fig.
1C). This effect was proportional
to
the amount of the EGF receptor-v-ErbB construct introduced
into the
cells and was observed with either the wild-type T3R
construct in the
absence of T3 hormone or a mutant of T3R (T3R-vA)
that is unable to
bind T3 hormone (Fig.
1C). The inhibitory effects
of EGF receptor
signaling on the SMRT-T3R interaction were not
limited to the v-ErbB
per se and could also be mediated by the
wild-type EGF receptor in
response to appropriate EGF receptor
ligands (data not
shown).
The effects of EGF receptor signaling in the two-hybrid assay system
appeared to reflect true inhibition of the SMRT-T3R interaction
itself:
v-ErbB had little or no effect on basal reporter expression
if either
(or both) of the GAL4DBD-SMRT or GAL4AD-T3R fusions
was omitted from
the transfection (Fig.
1C). Similarly, the introduction
of v-ErbB had
no effect on the expression of a

-galactosidase
reporter lacking
GAL4 binding sites and used as an internal control
(data not shown).
Therefore, the effects of v-ErbB in the two-hybrid
assay are unlikely
to be mediated by nonspecific inhibition of
the reporter promoter
itself or by a decrease in the stability
or the enzymatic activity of
the luciferase protein. To exclude
the possibility that v-ErbB
inhibited the expression or function
of the GAL4DBD or GAL4AD moieties,
rather than that it interfered
with the SMRT-T3R interaction itself, we
assayed the effects of
v-ErbB on the two-hybrid interaction between T3R
and RXRs. RXRs
are heterodimer partners for T3Rs, and the two receptor
classes
can physically associate in vitro and in vivo (
37).
In our mammalian
two-hybrid system, T3R exhibited a strong interaction
with RXRs
which was altered only slightly by cointroduction of v-ErbB,
in
clear contrast to the potent v-ErbB-mediated inhibition observed
for
the T3R-SMRT interaction (Fig.
1C). These results indicate
that the
interaction between SMRT and T3R is specifically inhibited
by
cointroduction of an activated EGF receptor-v-ErbB
construct.
SMRT function is inhibited by a MAPKKK signaling pathway.
The
EGF receptor can operate through a diverse array of downstream signal
regulators (13, 25, 60, 61, 66, 68). To evaluate which of
these downstream effectors might be responsible for the inhibition
exerted by the EGF receptor on SMRT function, we tested a variety of
candidate transducers and/or inhibitors of downstream signaling. A
specific chemical inhibitor of the tyrosine kinase activity of the EGF
receptor, AG1478 (34), abolished the effects of v-ErbB on
the SMRT-T3R interaction, suggesting that the kinase activity of the
EGF receptor-v-ErbB construct was essential for mediating the
inhibitory phenotype (Fig. 2A). Significantly, the introduction of a v-Ras expression vector mimicked the inhibitory effects of v-ErbB on the interaction of T3R and SMRT,
although not as strongly as did v-ErbB itself (Fig. 2B). In contrast,
treatment of CV-1 cells with moderate levels of cyclic AMP or phorbol
esters (inducers of protein kinase C) or introduction of an expression
vector for the catalytic subunit of phosphatidylinositol 3-kinase
failed to inhibit the T3R-SMRT interaction (Fig. 2B).

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FIG. 2.
Inhibition of the mammalian two-hybrid interaction
between SMRT and T3R by various signal transducers. (A) Effect of
tyrphostin AG1478 on the ability of v-ErbB to inhibit the SMRT-T3R
two-hybrid interaction. The GAL4DBD-SMRT fusion, the GAL4AD-T3R
fusion, and the GAL4-17-mer luciferase reporter were introduced into
CV-1 cells in the presence (+) or absence ( ) of the pSG5-v-ErbB
expression plasmid, as indicated below the panel. The cells were
subsequently incubated in the presence (+) or absence ( ) of 30 nM
tyrphostin AG1478, and the relative luciferase activity was determined.
(B) Effects of different signal transducers on the two-hybrid
interaction between SMRT and T3R-vA. CV-1 cells were transfected as
described in panel A with GAL4DBD-SMRT and GAL4AD-T3R-vA. The cells
were not treated (None), were treated with 10 µM 8-bromo-cyclic AMP
(cAMP) or 5 ng of phorbol-12-myristate-13 acetate (PMA) per ml, or were
cotransfected with expression plasmids for v-ErbB, v-Ras, v-Raf, or the
p110 catalytic subunit of phosphatidylinositol 3-kinase (PI3-K), as
indicated below the panel. The relative luciferase activity was
subsequently determined. The average and standard deviation of
duplicate experiments are presented.
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|
Ras is believed to operate primarily through the ability to bind to and
activate MAPKKKs, such as Raf and MEKK-1 (Fig.
3)
(
13,
25,
54,
60). Although
Raf is a downstream effector
of Ras signaling in many contexts,
introduction of an activated
Raf allele into our experimental system
had little or no effect
on the SMRT-T3R interaction (Fig.
2B). However,
MEKK-1 also can
serve as a downstream mediator of Ras function
(
13,
14,
25,
38,
48,
50,
54,
60), and introduction of either
a wild-type
or a constitutively activated allele of MEKK-1 into the
two-hybrid
assay resulted in very strong inhibition of the SMRT-T3R
interaction
(Fig.
4A). Similarly,
induction of endogenous MEKK-1 activity
with anisomycin also resulted
in inhibition of the SMRT-T3R interaction
(data not shown), whereas
introduction of a dominant negative
MEKK-1 allele into CV-1 cells
actually slightly enhanced the SMRT-T3R
interaction (Fig.
4A).
Introduction of MEKK-1 had little or no
effect on the two-hybrid
interaction of T3R with RXRs, indicating
that the inhibitory actions of
MEKK-1 were specific for the SMRT-T3R
interaction and did not represent
a nonspecific or indirect action
of MEKK-1 in the two-hybrid assay
system itself (Fig.
4A).

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FIG. 3.
Schematic of proposed MAPKKK signal transduction
pathways. The cascades include at least three major "modules," each
of which includes MAPKKKs (such as Raf, MEKK-1, or
mixed-lineage kinase [MLK]), MAPKKs (such as MEK-1/2, SEK-1, MAPKK-3,
MKK-6, or MKK-7), and MAPKs (such as ERK-1, SAPK- -Jun N-terminal
kinase [JNK], or p38). Upstream signals that activate these modules
include the EGF receptor (EGFR), tumor necrosis factor (TNF ), UV
light, and bacterial lipopolysaccharide (LPS). The major pathways are
indicated by solid lines; broken lines indicate cross talk that can
occur, at least under certain circumstances, between modules.
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FIG. 4.
Effect of MEKK-1 signaling on transcriptional
repression and T3R interactions with SMRT. (A) Inhibition by MEKK-1 of
the two-hybrid interaction between SMRT and T3R . The effect of
various signal transducers on the two-hybrid interaction between SMRT
and T3R was tested as described in the legend to Fig. 1C by
comparing an empty expression vector (None), a v-ErbB expression
plasmid (v-ErbB), a full-length MEKK-1 expression plasmid (MEKK1), or a
dominant negative MEKK-1 (residues 817 to 1493) expression plasmid
(MEKK1 DN), as indicated below the panel. (B) Effects of MEKK-1 and
MEK-1 on T3R -mediated repression of a TRE-luc reporter. The effect
of different signal transducers on the ability of T3R to
repress a TRE-driven promoter was tested as described in the legend to
Fig. 1A. Expression plasmids for full-length MEKK-1, dominant negative
MEKK-1, MEK-1, or dominant negative MEK-1 (MEK1 DN) were compared to an
equivalent empty expression vector (None), as indicated below the
panel. TRE reporter activity was assayed only in the absence of T3.
(Inset) Effect of dominant negative MEKK-1 on the ability of v-ErbB to
counteract T3R repression. Various combinations of expression plasmids
for T3R , v-ErbB, and dominant negative MEKK-1 were transfected into
CV-1 cells, as indicated below the inset panel, and the relative
luciferase activity was determined. Fold repression was calculated
relative to the basal levels of reporter gene
expression in the absence of T3R . (C) Effects of EGF receptor
and MEKK-1 signaling on transcriptional repression by PLZF. Various
amounts of a GAL4DBD-PLZF fusion construct were introduced into CV-1
cells together with the GAL4-17-mer luciferase reporter and
expression plasmids for v-ErbB or full-length MEKK-1. Relative
luciferase activity was determined by normalization to an internal
pCH110 -galactosidase control; fold repression was calculated
relative to the basal level of luciferase expression observed in the
absence of the PLZF construct. The average and standard deviation of
duplicate experiments are presented.
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This ability of MEKK-1 to inhibit the interaction of SMRT with T3R in a
two-hybrid assay was paralleled by the ability of
MEKK-1 to abrogate
T3R-mediated transcriptional repression (Fig.
4B). As previously noted,
T3R in the absence of hormone repressed
a reporter gene bearing a TRE
(Fig.
1B). Cointroduction of functional
MEKK-1 reversed this
repression, thereby mimicking the effects
of v-ErbB (Fig.
4B).
Conversely, introduction of a dominant negative
MEKK-1 allele failed to
reverse repression when introduced on
its own and partially
counteracted the inhibitory effects of v-ErbB
when cointroduced
together with the v-ErbB construct (Fig.
4B,
inset). Taken as a whole,
these results suggest that MEKK-1 is
able to operate downstream of the
EGF receptor to strongly inhibit
both the interaction of the SMRT
corepressor with T3R and the
ability of T3R to function as a
transcriptional
repressor.
MEK-1, an MAPKK, mimics some of the actions of MEKK-1, but a
variety of MAPKs do not.
We next explored which effectors
might be able to operate, in turn, downstream of MEKK-1 to regulate the
SMRT-T3R interaction. At least three parallel MAPKKK pathways
have been identified for metazoans; although some signaling is pathway
specific, there also exists detectable cross talk between the different
kinase hierarchies under certain experimental conditions (Fig. 3).
Intriguingly, the introduction of SEK-1, an MAPKK that in many contexts
functions immediately downstream of MEKK-1, had no significant effect
in our SMRT-T3R interaction assay (Fig.
5A). In contrast, the introduction of
MEK-1, a second form of MAPKK that has also been reported to operate
downstream of MEKK-1 (4, 14, 54, 77, 78), resulted in
inhibition of both the SMRT-T3R two-hybrid interaction and T3R-mediated
transcriptional repression (Fig. 4B and 5A). Consistent with these
results, MEK-1 activity, but not SEK-1 activity, was enhanced in CV-1
cells in response to the introduction of MEKK-1 (data not shown). Also
consistent with a role for MEK-1 in SMRT regulation, U0126 (a specific
inhibitor of MEK-1 and MEK-2 [15]) counteracted the
effects of MEK-1 in our transfection experiments (Fig. 5B). Notably,
however, MEK-1 was less efficient at inhibiting the SMRT-T3R
interaction (and T3R-mediated repression) than was MEKK-1 (Fig. 4B and
5A), and the counteracting effects of U0126 were much more pronounced
for MEK-1 than for MEKK-1 (Fig. 5B). Taken as a whole, these results
suggest that some, but not all, of the effects of MEKK-1 on the
SMRT-T3R interaction may be mediated by the downstream kinase MEK-1.

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FIG. 5.
Effects of different components of the MAPKKK cascade on
the ability of SMRT to interact with T3R or PLZF. (A) Effects of
different MAPKKK components on the mammalian two-hybrid interaction
between SMRT and T3R . The two-hybrid protocol used in Fig. 2B was
used but with expression vectors for v-ErbB, full-length MEKK-1, MEK-1,
ERK-1, SEK-1, SAPK- , or p38 or an empty vector, as indicated below
the panel. (B) Effect of a MEK-1 inhibitor, U0126, on the SMRT-T3R
interaction. The effects of MEKK-1 and MEK-1 on the SMRT-T3R
two-hybrid interaction were tested as described for panel A, but in the
absence ( ) or presence (+) of 15 µM U0126. (C) Effects of MEKK-1
and MEK-1 on the mammalian two-hybrid interaction between SMRT and
PLZF. The same protocol as that used in Fig. 2B was repeated but with
either an empty GAL4AD plasmid or a GAL4AD-PLZF fusion, together with
the GAL4DBD-SMRT construct previously described. The transfections were
performed in the presence of 100 ng of empty vector or an expression
vector for v-ErbB, MEKK-1, or MEK-1, as indicated below the panel. The
data represent the average and standard deviation of duplicate
experiments.
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To extend these results, we examined the effects of MEKK-1 and MEK-1 on
the ability of SMRT to interact with PLZF, a hormone-independent
transcriptional repressor. As previously noted (
20), the
ability
of SMRT to interact with PLZF in our two-hybrid system was
strongly
inhibited by cointroduction of EGF receptor-v-ErbB (Fig.
5C).
Significantly, the introduction of MEKK-1 or MEK-1 also interfered
with
the SMRT-PLZF interaction (Fig.
5C) and with PLZF-mediated
repression
(Fig.
4C). We conclude that these components of the
MAPKKK cascade
mediate strong inhibition of the ability of SMRT
to interact with a
variety of its transcription factor
partners.
In many regulatory pathways, the actions of MEKK-1 and MEK-1 are
mediated, in turn, by MAPKs that operate still lower in the
kinase
cascade (
13,
25,
60). However, overexpression of
three
different MAPKs, ERK-1, SAPK-

, and p38, separately or in
combination, had little or no observable effect on the T3R-SMRT
interaction in our two-hybrid system and no observable effect
on
T3R-mediated repression (Fig.
5A and data not shown). We conclude
that
the effects of MEKK-1 and MEK-1 on the T3R-SMRT interaction
are not
manifested through the actions of these downstream MAPKs;
the same
conclusion was obtained from our analysis of the sites
of
phosphorylation of SMRT (see
below).
The SMRT protein is phosphorylated in vivo and in vitro by multiple
components of the MAPKKK cascade.
To better understand the impact
of MEKK-1 signaling on the T3R-SMRT interaction, we next investigated
if either T3R or SMRT was modified by components of the MAPKKK signal
cascade. No indication of posttranslational modification of T3R, as
evidenced by a change in the apparent molecular weight of the nuclear
receptor, was observed in response to MEKK-1 or MEK-1 expression (data
not shown). In contrast, the introduction of MEKK-1 into transfected
cells led to a detectable change in the apparent mobility of the SMRT corepressor, as determined by Western blot analysis (Fig.
6A). A more modest shift in the mobility
of SMRT was also observed in response to the introduction of MEK-1
(Fig. 6A). The changes in the mobility of the SMRT protein in response
to MEKK-1 and MEK-1 were also observed upon introduction of v-ErbB or
v-Ras and by treatment of the cells with EGF receptor agonists, such as
transforming growth factor
(TGF-
), and were particularly evident
when corepressor constructs limited to the SMRT C-terminal receptor
interaction domain (RID) were used (Fig. 6A and B). The alterations in
SMRT mobility were reversed by treatment of the corepressor with
alkaline phosphatase and were not observed when kinase-defective
dominant negative mutants of either MEK-1 or MEKK-1 were used,
indicating that these mobility shifts were likely due to the
phosphorylation of SMRT (Fig. 6C).

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FIG. 6.
Effects of different signal effectors and transducers on
the electrophoretic mobility and phosphorylation of SMRT in transfected
cells. (A) Alterations in the electrophoretic mobility of SMRT in CV-1
cells in response to MEKK-1, MEK-1, or TGF- . CV-1 cells were
transfected with pCMV-SMRT and either an empty vector (None) or an
expression vector for full-length MEKK-1 or for activated MEK-1 or were
treated with 100 ng of TGF- per ml, as indicated above the panel.
The cells were subsequently lysed, and the extracts were resolved by
SDS-PAGE and analyzed by immunoblotting using antibody specific for the
SMRT protein. The mobility of the SMRT protein in the absence of
treatment is indicated by the broken line. (B) Alterations in the
electrophoretic mobility of an SMRT C-terminal polypeptide in CV-1
cells in response to different components of the MAPKKK cascades.
pCMV-SMRT-C (representing SMRT amino acids 751 to 1495) was introduced
into CV-1 cells, together with the various expression vectors indicated
above the panel. Whole-cell lysates were subsequently resolved by
SDS-PAGE and visualized by immunoblotting as described for panel A. (C)
Reversal by alkaline phosphatase of the alterations in SMRT
electrophoretic mobility. CV-1 cells were transfected with the
pCMV-SMRT-C construct and the various expression vectors indicated
above the panel. Whole-cell lysates were prepared and either mock
treated ( ) or incubated with 0.5 U of calf intestinal alkaline
phosphatase (CIAP) (+) for 30 min prior to analysis by SDS-PAGE and
immunoblotting.
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To identify the precise nature of the protein kinase(s) responsible for
these modifications, we tested the ability of SMRT
to be phosphorylated
in vitro by purified components of the MAPKKK
cascade (Fig.
7). Significantly, GST-SMRT protein
fusions were
phosphorylated in vitro by a variety of recombinant MEKK-1
preparations,
including a MEKK-1 preparation isolated by use of a
His
6 tag from
recombinant baculovirus-infected insect cells
(Fig.
7A) and MEKK-1
purified from recombinant
E. coli (Fig.
7B). Phosphorylation of
SEK-1, a known substrate for MEKK-1, is shown
for comparison (Fig.
7C). A GST fusion protein restricted to the
C-terminal domain
of SMRT (amino acids 1291 to 1495) was also
phosphorylated by
the recombinant MEKK-1 preparations, whereas more
N-terminal SMRT
domains and the GST protein itself were not (Fig.
7A
and B). Apparently
identical results were obtained when the kinase
assay was performed
with full-length MEKK-1 and full-length SMRT or
SMRT C-terminal
domain proteins immunopurified from suitably
transfected mammalian
cells (Fig.
7D). Intriguingly, activated (but not
unactivated)
MEK-1, either purified as a recombinant protein from
bacteria
or immunoenriched from transfected mammalian cells, could also
phosphorylate SMRT in vitro, with the major site(s) of MEK-1
phosphorylation
also mapping to a region within amino acids 1291 to
1495 of the
corepressor (Fig.
7B). None of our GST-SMRT constructs was
phosphorylated
in the absence of an exogenous kinase (Fig.
7). We
conclude that
SMRT appears to be a direct substrate for phosphorylation
by both
MEKK-1 and MEK-1, although presumably at distinct sites, and
that
the principal phosphorylation sites map within the C-terminal
RID
of the corepressor.

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FIG. 7.
Phosphorylation of SMRT in vitro and association of SMRT
with MEKK-1 and MEK-1 in vivo. (A) In vitro kinase assay of SMRT using
recombinant MEKK-1 purified from infected Sf9 insect cells. GST fusion
proteins representing different portions of SMRT, as indicated above
the panel, were synthesized in E. coli, immobilized on
glutathione-agarose, and incubated with [ -32P]ATP in
the presence (right panel) or absence (left panel) of
His6-MEKK-1 purified from recombinant baculovirus-infected
Sf9 cells. Phosphorylated SMRT proteins are indicated by arrowheads.
(B) In vitro kinase assay of SMRT protein derivatives using recombinant
MEKK-1 and MEK-1 purified from E. coli. The same protocol as
that used in Fig. 7A was used, except that bacterially expressed MEKK-1
or MEK-1 was used in the kinase assay. Phosphorylated SMRT proteins are
indicated by arrowheads. (C) Comparison of MEKK-1 phosphorylation of
SMRT and of SEK-1. GST-SMRT or GST-SEK-1 protein derivatives were
incubated with [ -32P]ATP in the presence (+) or
absence ( ) of recombinant murine MEKK-1 (residues 817 to 1493)
purified from E. coli, as indicated below the autoradiogram.
Incubations were performed with 20 mM morpholinepropanesulfonic acid
(MOPS) (pH 7.2), 25 mM 2-glycerolphosphate, 5 mM EGTA, 1 mM DTT, 1 mM
sodium vanadate, and 15 mM MgCl2. The locations of the
phosphorylated SMRT and SEK-1 proteins are indicated by arrowheads. (D)
In vitro kinase assay of SMRT proteins using full-length MEKK-1
isolated from COS-1 cells. The kinase assay was performed with SMRT
proteins and full-length MEKK-1 immunopurified from transfected COS-1
cells rather than the recombinant proteins used in panel C. The
positions of the phosphorylated SMRT derivatives are indicated by
arrowheads, as is the position of MEKK-1, which is autophosphorylated
in these assays. C-term, C terminus. (E) Physical association of SMRT
with MEKK-1 and MEK-1 in mammalian cells. COS-1 cells were transfected
with pCMV-SMRT and pCMV-HA-MEK-1 in the presence or absence of
pCMV-FLAG-MEKK-1, as indicated below the panel. The cells were lysed,
and the lysates were analyzed directly by SDS-PAGE and immunoblotting
(left panel). Alternatively, the lysates were immunoprecipitated (IP)
with anti-FLAG antibody M2, and the resulting immunoprecipitates were
analyzed by SDS-PAGE and immunoblotting (right panel). The antisera
used to visualize each immunoblot are indicated on the left.
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Although overexpression of the MAPKs ERK-1, p38, and SAPK-

had no
effect on the T3R-SMRT interaction in our two-hybrid assay,
the SMRT
protein was nonetheless capable of being phosphorylated
by MAPKs both
in vitro and in vivo. SMRT was shifted in mobility
by coexpression of
the MAPK ERK-1 or p38 in vivo, and two domains
of SMRT were
phosphorylated by purified MAPK in vitro: (i) amino
acids 566 to 1075, overlapping the silencing domain of SMRT, and
(ii) amino acids 1056 to
1291, representing the RID of SMRT (Fig.
6B and
8A and data not shown). We therefore
wished to confirm
that the phosphorylation of SMRT by these MAPKs was
not involved
in mediating the MEKK-1 inhibition phenotype. The
inhibitory effects
of MEKK-1 signaling on the two-hybrid interaction
were observed
with a SMRT construct limited to the C-terminal half of
SMRT,
so we focused our analysis on the MAPK sites within the
corresponding
SMRT region (amino acids 1055 to 1291). This region has
five potential
recognition sites for MAPK which we altered,
individually or in
combination, to alanines and tested for their
effects on MAPK
phosphorylation (Fig.
8B). Phosphorylation of the SMRT
RID by
ERK-2 was inhibited by over 75% by an S1239A substitution and
virtually eliminated by an S1095A/S1239A double substitution;
the
remaining alanine substitution mutant proteins were phosphorylated
at
levels comparable to that of the wild-type SMRT protein (Fig.
8B).
These results implicate serine 1095 and serine 1239 of SMRT
as the
primary sites of phosphorylation of the SMRT C terminus
by this MAPK.

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FIG. 8.
Phosphorylation of SMRT in vitro by MAPK. (A)
Phosphorylation of SMRT by ERK-2. GST fusions representing different
regions of SMRT were incubated with [ -32P]ATP and
either no kinase (left panel) or purified ERK-2 (right panel). The
proteins were resolved by SDS-PAGE and visualized by PhosphorImager
analysis. Arrows indicate phosphorylated GST-SMRT derivatives. A
schematic below the gel depicts the regions of SMRT represented by the
different GST fusions. Inter., interaction domains. (B) Effects of
alanine substitutions on the phosphorylation of SMRT by ERK-2.
Different serine codons were converted into alanines by site-directed
mutagenesis, as indicated below the panel. The different mutants were
expressed as GST-SMRT (residues 1291 to 1495) fusion proteins and were
tested for the ability to be phosphorylated by ERK-2 in vitro as
described for panel A. The amount of 32P incorporated into
each mutant or wild-type protein was quantified by PhosphorImager
analysis. (C) Response of an MAPK-deficient mutant of SMRT to MEKK-1
and MEK-1 signaling. The mammalian two-hybrid experiment shown in Fig.
5A was repeated with pSG5-GAL4AD-T3R and either pSG5-GAL4DBD-SMRT
(wild type) (left panel) or pSG5-GAL4DBD-SMRT bearing a double
substitution (S1095A/S1239A) (right panel).
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Although unable to be phosphorylated by MAPK, the 1095A/1239A double
mutant of SMRT was fully susceptible to inhibition by
MEKK-1 and MEK-1
in the two-hybrid T3R interaction assay (Fig.
8C and data not shown).
In addition, the mobility shift observed
for SMRT in transfected cells
in response to MEKK-1 and MEK-1
signaling was not eliminated by
introduction of the 1095A/1239A
double mutation, indicating that this
mutant remains a substrate
for MEKK-1 and MEK-1 (data not shown). We
conclude that although
MAPKs are able to phosphorylate SMRT, they do
not appear to be
the principal effectors by which the inhibitory
effects of MEKK-1
or MEK-1 on the SMRT-T3R interaction were manifested
in our two-hybrid
assay. Instead, inhibition of SMRT function was
closely correlated
with phosphorylation of the corepressor by MEKK-1
and, to a lesser
extent, MEK-1.
SMRT can be isolated from cells in the form of a complex with
MEKK-1 and MEK-1.
The individual kinases within certain MAPKKK
cascades appear able to associate together to form a physical complex
that can, in turn, bind to and phosphorylate substrate
polypeptides (reviewed in references 53 and
70). To test if a similar physical complex might
be involved with the phosphorylation of SMRT, we cotransfected cells with expression vectors for SMRT and for a FLAG-tagged
MEKK-1 or a hemagglutinin (HA)-tagged MEK-1 construct.
Immunoprecipitates of FLAG-tagged MEKK-1 or HA-tagged MEK-1
contained high levels of associated SMRT protein in Western analysis,
whereas little or no SMRT was immunoprecipitated by anti-FLAG
antibodies in the absence of a tagged MEKK-1 construct (Fig. 7E).
Identical results were observed in immunoprecipitations of full-length,
rather than epitope-tagged, MEKK-1 (data not shown). Notably,
FLAG-tagged MEK-1 was also detected in immunoprecipitates of
FLAG-tagged MEKK-1 and visa versa (e.g., Fig. 7E). Taken as a whole,
our results indicate that MEKK-1 and MEK-1 can be found in a physical
complex in cells and that this complex itself appears able to bind to and phosphorylate the SMRT corepressor.
Phosphorylation by MEKK-1 or by MEK-1 interferes with the ability
of SMRT to bind to nuclear hormone receptors in vitro.
We next
determined if the inhibitory effects of MEKK-1 and MEK-1 on the
interaction between SMRT and T3R in our two-hybrid system were mimicked
in vitro. We tested the ability of radiolabeled SMRT, synthesized by in
vitro translation, to bind to a GST-T3R fusion protein synthesized in
bacteria. A strong interaction was observed between SMRT and the
GST-T3R polypeptide in the absence of exogenous kinases (Fig.
9A). Preincubation of the SMRT protein with either purified MEKK-1 or purified MEK-1 led to more than 66%
inhibition of the ability of the corepressor to bind to the GST-T3R
construct (Fig. 9A). The inhibitory effects of SMRT phosphorylation in
vitro may be even greater than this 66% value suggests: the SMRT
protein population that bound to the GST-T3R matrix after kinase
treatment migrated with a mobility characteristic of unphosphorylated SMRT, suggesting that it may have escaped being phosphorylated by the
kinase or was dephosphorylated during subsequent incubations (data not
shown). Incubation of SMRT with purified MAPKs, such as ERK-2, had no
effect on the ability of the corepressor to bind to T3R (Fig. 9B). We
conclude that phosphorylation of SMRT by MEKK-1 or MEK-1 specifically
interferes with the ability of the corepressor to bind to nuclear
receptors, such as T3R, in vitro as well as in vivo.

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FIG. 9.
Inhibition of the SMRT-T3R interaction in vitro by
phosphorylation by MEKK-1 and MEK-1. (A) Inhibition by MEKK-1 or MEK-1
of the ability of SMRT to bind to GST-T3R in vitro. Nonrecombinant
GST or a GST-T3R fusion protein was synthesized in E. coli and immobilized on glutathione-agarose. The immobilized GST
proteins were then incubated with 35S-labeled SMRT protein
that had been previously incubated without kinase, with bacterially
produced MEKK-1, or with bacterially produced, activated MEK-1, as
indicated below the panel. Radiolabeled SMRT remaining bound to the
glutathione-agarose after extensive washing was eluted with free
glutathione and was resolved by SDS-PAGE. The amount of bound SMRT was
quantified by PhosphorImager analysis and is presented as a
percentage of the total input for each binding reaction. The data
represent the average and range of duplicate experiments. (B) Lack of
an effect of ERK-2 phosphorylation on the ability of SMRT to bind to
GST-T3R in vitro. An experiment similar to that shown in panel A was
performed but with purified ERK-2 instead of MEKK-1 or MEK-1.
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MEKK-1 signaling alters the subcellular localization of the SMRT
protein.
We next examined the effects of the MEKK-1 cascade on the
subcellular distribution of SMRT. N-CoR and SMRT are nuclear proteins. In agreement with prior observations on native SMRT (57), a GFP fusion of SMRT was located virtually exclusively in the nucleus of
unstimulated transfected cells, forming a punctate pattern of bright
fluorescent spots superimposed on a more diffuse fluorescent nucleoplasm (Fig. 10A). Cointroduction
of a v-ErbB, MEKK-1, or MEK-1 expression vector into these cells led to
a change in the GFP-SMRT pattern, manifested as coalescence of the
punctate spots into a smaller number of larger dots per nucleus. In
many cells, the GFP-SMRT signal also shifted out of the nucleus into a
perinuclear or cytoplasmic fluorescence pattern; this effect was most
evident in response to MEKK-1 and was not observed with a
kinase-defective MEKK-1 mutant (Fig. 10A and data not shown). No
change in the morphology or integrity of the nuclei themselves was
observed. The introduction of MAPKs, such as ERK-1 or p38, had no
observable effect on the subcellular distribution of SMRT. The
1095A/1239A mutant construct of SMRT, which is defective for MAPK
phosphorylation in vitro, underwent the same change in subcellular
distribution in response to MEKK-1 signaling as did the wild-type SMRT
protein (data not shown).


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FIG. 10.
Effect of transient MEKK-1 expression on subcellular
localization of SMRT proteins. (A) Alteration of subcellular
localization of GFP-SMRT protein by MEKK-1 and MEK-1 signaling. CV-1
cells were transfected with pCMV-GFP-SMRT together with an empty
expression plasmid or were cotransfected with expression vectors for
v-ErbB, MEKK-1, or MEK-1. The GFP signal was subsequently visualized by
confocal microscopy. Representative cell fields are shown at different
magnifications. Arrows indicate the nuclear envelope. (B) Biochemical
subcellular fractionation of SMRT proteins. pCMV-SMRT-C was introduced
into CV-1 cells with either an empty vector (None) or v-ErbB,
full-length MEKK-1, MEK-1, or v-Ras expression vectors, as
indicated above the immunoblot. The cells were harvested and separated
into nuclear and cytoplasmic fractions as described in Materials and
Methods. SMRT proteins were not detected in the cytoplasmic fraction
when coexpressed with p38 or SEK-1 (data not shown).
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We confirmed these results using a biochemical subcellular
fractionation procedure (Fig.
10B). Consistent with the GFP fusion
data, in the absence of MEKK-1 signaling all of the SMRT protein
detected by Western analysis was found in the nuclear fraction,
whereas
the introduction of MEKK-1 or, to a somewhat lesser extent,
MEK-1
resulted in a significant redistribution of the SMRT protein
from the
nuclear to the cytoplasmic fraction (Fig.
10B). V-ErbB
and v-Ras
overexpression also led to a redistribution of SMRT
into the
cytoplasmic fraction (Fig.
10B). We conclude that MEKK-1
signaling can
result in a change in the subcellular distribution
of the SMRT protein
from an exclusively nuclear compartment to
a more perinuclear and
cytoplasmic
distribution.
 |
DISCUSSION |
A MAPKKK cascade acts to inhibit SMRT function.
The ability of
nuclear hormone receptors and of nonreceptor transcription factors,
such as PLZF, to repress transcription is strongly counteracted by the
EGF receptor signal transduction pathway (20). This
inhibition of repression by EGF receptor signaling appears to be due to
an inhibition of the ability of the SMRT corepressor to physically
interact with the transcription factor partner, as observed both in a
mammalian two-hybrid assay and by coprecipitation analyses (20,
30). Given that EGF receptor signaling had comparable inhibitory
effects on the ability of SMRT to interact with T3R, RAR, and PLZF, we
had previously proposed that the corepressor itself was likely to
represent the common target through which these EGF receptor-initiated
events manifest their inhibitory effects (20).
In the current study, we found evidence that the SMRT corepressor is
phosphorylated in response to EGF receptor signaling,
at least in part
in response to a MAPKKK pathway operating downstream
of the EGF
receptor, whereas T3R is not; this corepressor phosphorylation
closely
correlates with and may mediate the inhibition of SMRT
function
reported previously. Intriguingly, the SMRT corepressor
is subject to
phosphorylation by kinases operating at a variety
of levels
of the MAPKKK regulatory cascade, including MEKK-1,
MEK-1,
and MAPKs, such as ERK-1, ERK-2, and p38. The strongest
inhibitory
effects on SMRT function are associated with the actions
of MEKK-1,
with overexpression of MEKK-1 resulting in both a dramatic
decrease in
the ability of SMRT to interact with its transcription
factor partners
in a two-hybrid interaction assay and a parallel
inhibition of
transcriptional repression by these transcription
factors. MEK-1, an
MAPKK that operates at a level below that of
MEKK-1, mimicked some, but
not all, of the actions of MEKK-1.
In contrast, phosphorylation of SMRT
by several different MAPKs
had no detectable effect on corepressor
function in the assays
described here (see
below).
MEKK-1 signaling is coupled to that of growth factor receptors, such as
the EGF receptor, by the actions of Ras (reviewed
in references
13,
25,
54, and
60). Consistent
with the
proposal that MEKK-1 can operate downstream of the EGF
receptor
to inhibit SMRT, Ras expression also inhibits SMRT function,
although
less efficiently than does the expression of MEKK-1. MEKK-1
also
plays an important role in responding to signals of cell stress
(
13,
54,
60), and induction of cell stress with anisomycin
leads to strong inhibition of both the SMRT-T3R interaction and
SMRT-mediated repression, consistent with our proposed role of
MEKK-1
as a negative modulator of SMRT function. MEKK-1 appears
to be
relatively specific in its ability to inhibit SMRT; Raf,
a second
MAPKKK that also operates downstream of Ras, had no detectable
effect
on SMRT function in our
experiments.
SMRT appears to be a direct substrate for MEKK-1-mediated
phosphorylation.
The ability of MEKK-1-mediated signaling to
inhibit SMRT function was closely paralleled by an increase in the
overall level of phosphorylation of the SMRT protein, as manifested by
changes in the mobility of SMRT on Western blots that could be reversed with phosphatase treatment. SMRT could also be phosphorylated in vitro
using a variety of preparations of purified or enriched MEKK-1.
These results indicate that SMRT is phosphorylated either by
MEKK-1 itself or possibly by a tightly associated kinase that copurifies with MEKK-1. We favor the former hypothesis for several reasons. (i) MEKK-1 could phosphorylate SMRT in vitro when both the
kinase and the substrate were purified as recombinant proteins from
E. coli, eliminating the possibility that these preparations were contaminated with other eukaryotic kinases. (ii) Kinase-defective mutants of MEKK-1 were impaired in the ability to phosphorylate SMRT
(unpublished observations). (iii) In common with many previously identified substrates of MEKK-1, SMRT could be isolated in the form of
a physical complex with MEKK-1. We have mapped a major site of
MEKK-1-mediated phosphorylation of SMRT in vitro to within the SMRT
RID; however, given that the substrate sequence specificity of MEKK-1
remains poorly understood, we have not yet defined the location of this
phosphorylation to a specific amino acid within this corepressor domain.
It might appear paradoxical that MEKK-1, thought to be largely a
cytoplasmic or an inner plasma membrane protein, is able
to
phosphorylate SMRT, a transcriptional modulator that functions
in the
nucleus. MEKK-1 may be able to access the nuclear compartment,
perhaps
transiently, during different stages of the cell cycle
(
14).
Alternatively, MEKK-1 may phosphorylate nascent SMRT after
its
synthesis on cytoplasmic ribosomes but before translocation
of the
corepressor into the nucleus. It may be relevant that the
actions of
MEKK-1 lead to an increased cytoplasmic localization
of SMRT, perhaps
further increasing its availability for modification
by this kinase. It
is intriguing in this regard that another target
of MEKK-1 regulation
is the NF-

B-I

B transcription factor complex,
which resides in
the cytoplasm in unstimulated cells. MEKK-1 can
phosphorylate the I

B
kinase, leading to phosphorylation of I

B
and release of NF-

B
(
32). MEKK-1 leads to nuclear translocation
and activation
of NF-

B, whereas we propose that MEKK-1 phosphorylation
of SMRT
leads to cytoplasmic retention and inactivation of the
corepressor.
SMRT is also a target for phosphorylation by a diverse array of
additional protein kinases.
As detailed above, SMRT
phosphorylation is increased in cells by the introduction of active
MEKK-1, and at least one component of this enhanced phosphorylation
appears to be due to direct phosphorylation by MEKK-1. However,
SMRT is also phosphorylated at distinct sites by MEK-1 and by MAPKs.
Phosphorylation of SMRT by MEK-1 can mimic some of the effects of
MEKK-1, although more weakly, whereas phosphorylation of SMRT by MAPKs
does not have any observable effect on the ability of SMRT either to
associate with nuclear receptors in vivo or in vitro or to mediate
transcriptional repression under the conditions tested. In addition to
the phosphorylation of SMRT by known components of the MAPKKK cascade,
we have also observed that SMRT is phosphorylated in vitro and in vivo
by casein kinase II and apparently by as-yet-unidentified kinases found
physically associated with SMRT in CV-1 cell lysates (unpublished observations).
It is intriguing that SMRT serves as a substrate for so many kinases
operating both within and without the known MAPKKK cascades.
Although
typically portrayed as acting in a linear and hierarchical
fashion, MAPKKK signal transduction cascades often operate at
multiple
levels. For example, as shown here for SMRT and as noted
elsewhere for
I

B kinase and Smad2 (
9,
32), MEKK-1 can function
not only
by transducing signals to kinases lower in the hierarchy,
such as MEK-1
and MAPKs, but also by itself phosphorylating important
substrates
directly. In addition, the different MAPKKK cascades
in eukaryotic
cells can exhibit considerable cross talk between
one another,
depending on the cell type and experimental conditions.
Thus, MEKK-1 is
an important transducer of the cell stress signal
but also appears to
respond to growth factor signals mediated
through Ras. Similarly,
although SEK-1 is believed to be the principal
MAPKK operating
downstream of MEKK-1, MEK-1 appears able to serve
this role in the CV-1
cell experiments described
here.
Mechanism of the inhibition of SMRT function by MEKK-1.
How
does MEKK-1 and MEK-1 signaling operate at the molecular level to
inhibit the interaction between SMRT and its transcription factor
partners? Notably, the ability of SMRT to physically associate with T3R
in vitro is inhibited by incubation with either purified MEKK-1 or
purified MEK-1, suggesting that the phosphorylation of the corepressor
by these kinases can decrease the affinity of the corepressor for
transcription factors and may account, at least in part, for the
corresponding inhibition of the two-hybrid interaction observed in
cells. This hypothesis is consistent with the sites of MEKK-1 and MEK-1
phosphorylation within SMRT, which both map within the RID of the
corepressor. In contrast to this inhibition mediated by MEKK-1 and
MEK-1, the phosphorylation of SMRT in vitro by MAPKs did not detectably
affect the interaction between the corepressor and T3R.
However, the direct inhibitory effects of MEKK-1 and MEK-1
phosphorylation observed in vitro may not represent the only inhibitory
effect of the MAPKKK pathway on SMRT function. Significantly,
we
observed that overexpression of MEKK-1 or, to a lesser extent,
of MEK-1
led to relocalization of SMRT from an almost exclusively
nuclear
compartment to a perinuclear or cytoplasmic compartment
in transfected
cells. This relocalization of SMRT was observed
using a GFP-SMRT
immunofluorescence technique and, independently,
in biochemical
subcellular fractionation experiments. We propose
that in addition to
the direct inhibitory effect of MEKK-1 phosphorylation
on the
interaction of SMRT with its transcription factor partners
in vitro,
MAPKKK signaling also leads to a redistribution of SMRT
from the
nucleus to the cytoplasm of the cell. This change in
the subcellular
localization of SMRT may contribute, at least
in part, to the loss of
interaction of the corepressor and nuclear
transcription factors
observed in two-hybrid assays and the loss
of SMRT-mediated repression
seen in transcription
assays.
We do not yet understand the precise mechanisms by which MEKK-1 and
MEK-1 signaling leads to the alteration in subcellular
localization
observed for SMRT. This change in SMRT localization
may occur directly
in response to the phosphorylation of the corepressor
itself or
may be mediated by a more indirect mechanism, such as
an
MAPKKK-induced change in the nuclear export or import machinery
of the cell. We also do not yet know if the change in the subcellular
localization of SMRT represents a direct effect of the actions
of the
MAPKKK pathway or if the SMRT redistribution is a secondary
effect
occurring as a consequence of the loss of tethering of
the corepressor
to its transcription factor partners. We are currently
investigating
these issues in more
detail.
Protein kinases and nuclear hormone receptors
a convergence of
cell signaling pathways.
Many tantalizing links have been
established between the actions of protein kinases and those of nuclear
hormone receptors. For example, many nuclear hormone receptors are
themselves targets of phosphorylation by a variety of kinases, such as
protein kinase A, the cyclin-dependent kinases, DNA-dependent protein
kinases, and casein kinases; phosphorylation by these kinases can
either enhance or impair nuclear receptor function (reviewed in
references 6 to 8, 26, 56, and
69). Intriguingly, there appears to be a
particularly intimate series of interconnections between the functions
of the nuclear hormone receptors and MAPKKK regulatory cascades
(10, 16, 23, 24, 26, 27, 49, 56). Here we have shown that
the corepressor SMRT is also an important target of modification by a
diverse array of protein kinases, including components of the MAPKKK
cascades, and that at least some of these phosphorylation events can
disrupt SMRT function. It appears likely that the actions of
coactivators and corepressors are subject to similar forms of
posttranslational regulation (1, 42). Taken as a
whole, our results suggest that multiple interactions take place
between ligand-dependent and ligand-independent signal transduction
pathways and that these interactions operate at multiple levels
to generate both convergence and integration of different signals and
thus to generate the correct combinatorial regulation of target gene expression.
 |
ACKNOWLEDGMENTS |
We thank J. M. Bishop, R. M. Evans, L. Freedman, J. Kyriakis, M. Lazar, and T. Maniatis for generously providing molecular clones used in this research.
This work was supported by Public Health Service grants R37 CA-53394
and R01 DK-53528 from NIH.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Section of
Microbiology, University of California at Davis, One Shields Ave.,
Davis, CA 95616. Phone: (530) 752-3013. Fax: (530) 752-9014. E-mail: mlprivalsky{at}ucdavis.edu.
 |
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