Signaling by Tyrosine Kinases Negatively Regulates the Interaction between Transcription Factors and SMRT (Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor) Corepressor
Suk-Hyun Hong,
Chi-Wai Wong and
Martin L. Privalsky
Section of Microbiology Division of Biological Sciences
University of California at Davis Davis, California 95616
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ABSTRACT
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Nuclear hormone receptors are
hormone-regulated transcription factors that bind to specific sites
on DNA and modulate the expression of adjacent target genes. Many
nuclear hormone receptors display bimodal transcriptional properties;
thyroid hormone receptors, for example, typically repress target gene
expression in the absence of hormone, but activate target gene
expression in the presence of hormone. The ability to repress is
closely linked to the ability of the apo-receptor to physically bind to
auxiliary corepressor proteins denoted SMRT (silencing mediator of
retinoic acid and thyroid hormone receptor) and N-CoR (nuclear receptor
corepressor), which, in turn, help mediate the actual molecular events
involved in transcriptional silencing. We report here that repression
by thyroid hormone receptors can be regulated not only by cognate
hormone, but also by certain tyrosine kinase signal transduction
pathways, such as that represented by the epidermal growth
factor-receptor. Activation of tyrosine kinase signaling leads
to inhibition of T3R-mediated repression with
relatively little effect on activation. These effects appear to be
mediated by a kinase-initiated disruption of the ability of
T3R to interact with SMRT corepressor.
Intriguingly, tyrosine kinase signaling similarly disrupted the
interactions of SMRT with v-Erb A, with retinoic acid
receptors, and with PLZF, a nonreceptor transcriptional repressor. We
conclude that tyrosine kinase signaling exerts potentially important
regulatory effects on transcriptional silencing mediated by a variety
of transcription factors that operate through the SMRT corepressor
complex.
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INTRODUCTION
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Many crucial processes of vertebrate homeostasis, reproduction,
and differentiation are regulated by the actions of small, lipophilic
hormones (reviewed in Refs. 1, 2, 3, 4, 5, 6, 7). These lipophilic hormones include
the steroids, retinoids, and thyroid hormones are sensed by nuclear
hormone receptors that bind to cognate hormone and function as
ligand-regulated transcription factors (1, 2, 3, 4, 5, 6, 7). Each nuclear hormone
receptor binds to specific sites on the DNA genome and modulates the
expression of specific sets of target genes. Intriguingly, nuclear
hormone receptors can exert both positive and negative effects on
transcription. These bimodal transcriptional properties are manifested
through the ability of these receptors to associate with auxiliary
regulator proteins, denoted corepressors and coactivators, that mediate
the actual transcriptional response (8). For example, thyroid hormone
receptors (T3Rs) and retinoic acid receptors (RARs) can
function as transcriptional silencers in the absence of hormone, a
context in which these receptors bind to a class of corepressor
proteins denoted SMRT (silencing mediator of retinoic acid and thyroid
hormone receptor)/N-CoR (nuclear receptor corepressor) (also known as
TRAC and RIP13) (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21). Binding of hormone converts T3Rs
and RARs into strong transcriptional activators, a process that is
accomplished by the release of the SMRT/N-CoR corepressors and the
subsequent association of the receptors with a new set of proteins that
function as transcriptional coactivators (9, 10, 11, 12, 14, 18, 19, 22).
Thus, repression by nuclear hormone receptors involves a physical
recruitment of SMRT/N-CoR corepressor proteins to the target promoter.
The SMRT/N-CoR proteins can recruit, in turn, an ensemble of additional
polypeptides that includes mSin3A/B, histone deacetylases (HDAC-1 and
2), retinoblastoma protein-associated proteins 46 and 48, and several
other polypeptides of as-yet-unknown function (reviewed in Refs. 23, 24). It is this larger corepressor complex that is thought to be the
actual effector of transcriptional repression. Repression of gene
expression probably involves multiple mechanisms, including both
modifications of the chromatin template and inhibitory interactions
with the general transcriptional machinery itself (17, 23, 24).
Notably, although SMRT and N-CoR were initially identified as
corepressors for the nuclear hormone receptors, the different
components of the SMRT/N-CoR/Sin3/HDAC complex also appear to play a
key role in transcriptional silencing by a wide variety of nonreceptor
transcription factors, including Mad/Max, YY-1, PLZF, BCL-6, the
retinoblastoma gene product, and several yeast transcriptional
regulators (23, 24, 25, 26, 27, 28).
Nuclear hormone receptors appear to serve as an important molecular
nexus at which a variety of hormonal and nonhormonal signals converge
to generate a combinatorial regulation of target gene expression. The
actual transcriptional response in vivo is determined not
only by the hormone status, but also by the nature of the target
promoter, and by the actions of nonligand signal transduction pathways
in the cell (29, 30, 31, 32, 33, 34). Particularly interesting is the ability of
certain protein kinases to modulate, both negatively and positively,
nuclear hormone receptor function (reviewed in Refs. 31, 32, 33, 34). For
example, certain nonligand signal transduction pathways can induce
target gene activation, even in the absence of ligand, or can further
enhance the activation observed in the presence of hormone ligand
(e.g. Refs. 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45). In some cases these effects may be
mediated through posttranslational modifications of the receptor itself
(32, 35, 41, 44, 45, 46). In other contexts, however, the mechanism by
which a signal transduction pathway alters the regulatory properties of
the nuclear hormone receptor does not appear to involve a detectable
alteration in the receptor itself (36, 43, 47).
We wished to investigate whether the SMRT corepressor might represent a
previously unrecognized target for these nonligand signal transduction
pathways. We report here that activation of a cell surface tyrosine
kinase, the epidermal growth factor-receptor [EGF-R (48)] or its
constitutively activated allele, the v-Erb B oncogene (49),
can interfere with T3R-mediated repression with little or
no effect on T3R-mediated gene activation. This relief of
repression by tyrosine kinase signaling is manifested as a severe
inhibition of the interaction between T3R and SMRT
corepressor. Notably, the interactions between SMRT and RARs, or
between SMRT and PLZF (a nonreceptor transcriptional repressor), are
similarly strongly inhibited by activation of the
EGF-R/v-Erb B tyrosine kinase-signaling pathway. We
conclude that certain cell surface tyrosine kinases can mediate effects
on transcription by nuclear hormone receptors, and by POZ domain
proteins such as PLZF, by interfering with ability of the SMRT
corepressor complex to tether to these transcription factors.
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RESULTS
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Activation of a Tyrosine Kinase Signal Transduction Pathway
Interferes with T3R-Mediated Transcriptional
Repression in CV-1 Cells
We assayed the transcriptional properties of T3R by
transient transfections of CV-1 cells. The reporter gene was a
thymidine kinase promoter-luciferase (tk-luc) construct bearing
T3R-binding sites (i.e. thyroid hormone response
elements, or TREs). CV-1 cells possess very low levels of endogenous
T3Rs, and the basal expression of the TRE-tk-luc reporter exhibited
little or no response to thyroid hormone if no exogenous receptor was
introduced (Fig. 1
). Consistent with
previous work (19), cointroduction of T3R
into the CV-1
cells resulted in a repression of this reporter gene in the absence of
T3 hormone and in a stimulation of luciferase expression in
the presence of T3 hormone (Fig. 1A
). This bimodal hormone-dependent
activity required the presence of TRE sites in the reporter and was not
observed for a ß-galactosidase reporter, lacking TREs, employed as an
internal negative control (data not shown).
We next repeated these experiments, but with the cointroduction
of v-Erb B, a constitutively-activated derivative of the
host cell EGF-R; v-Erb B is a membrane-associated
tyrosine kinase that functions independently of EGF ligand and was
employed to uniformly and strongly activate the EGF-R signal
transduction pathway in the transfected cells (48, 49). Intriguingly,
the introduction of v-Erb B significantly impaired
T3R-mediated repression in the absence of T3
hormone, but had only a modest effect on T3R-mediated
activation in the presence of T3 hormone (Fig. 1
). In
contrast, introduction of v-Erb B in the absence of
T3R had little or no effect on the basal level of
expression of the TRE-tk-luc reporter, indicating that the primary
effect of v-Erb B was to counteract T3R
repression, rather than to stimulate reporter gene expression
independent of the actions of T3R.
A retrovirus-transduced, mutant version of a normal cellular
T3R, denoted v-Erb A, is in fact, encoded by the
same retrovirus as is v-Erb B, and the two oncoproteins
operate together in leukemogenesis (49). V-Erb A has lost
the ability to bind to T3 hormone and functions in many
contexts as a hormone-independent transcriptional repressor (50, 51).
Notably, cointroduction of v-Erb B interfered with
v-Erb A-mediated repression in a fashion similar to the
effects of v-Erb B on repression mediated by the wild-type
T3R (Fig. 1B
; the very modest change in reporter expression
observed on introduction of v-Erb B in the absence of
v-Erb A likely reflects small differences in the overall
physiology of the transfected cells in the presence and absence of the
tyrosine kinase). We conclude that the ability of v-Erb B to
reverse repression by T3R derivatives is independent of the
ability of the nuclear receptor itself to bind, or to respond directly,
to cognate T3 hormone.
Activation of the Tyrosine Kinase-Signaling Pathway Is Manifested
as Inhibition of the Interaction between T3R
and SMRT Corepressor
The abrogation of T3R-mediated repression by
v-Erb B suggested that the EGF-R signal transduction pathway
might be capable of altering the interaction between T3R
and SMRT corepressor. We first tested this possibility by use of a
mammalian two-hybrid interaction assay. For this assay, the
receptor-interaction domains of SMRT were fused to the DNA-binding
domain (DBD) of GAL4 (GAL4-DBD) and inserted into a mammalian
expression vector, pSG5. In parallel, relevant portions of the nuclear
hormone receptors were fused to the activation domain of GAL4 (GAL4-AD)
and placed into the same pSG5 vector. In this manner, interaction
between SMRT and receptor should lead to a functional reconstitution of
the GAL4 transcriptional activator, assayed as stimulation of a
GAL4(17-mer)-luciferase reporter, when all three constructs are
cointroduced into mammalian CV-1 cells (26).
Introduction of the GAL4DBD-SMRT fusion together with an "empty"
pGAL4-AD construct, or an empty GAL4DBD construct together with the
GAL4-AD-T3R
fusion, had little or no effect on
expression of the GAL4(17-mer)-luciferase reporter (Fig. 2A
). In contrast, cointroduction of both
constructs led to a strong stimulation of luciferase expression,
consistent with the known ability of SMRT and T3R to
strongly interact (Fig. 2A
). This SMRT/T3R interaction
assayed by the two-hybrid protocol displayed all of the properties
expected from prior in vitro and in vivo studies:
1) it was abolished by addition of thyroid hormone; 2) mutations of
T3R that are unable to interact with SMRT in
vitro (such as the P156R substitution) fail to function in the
mammalian two-hybrid assay; 3) deletions of SMRT that are defective for
receptor binding in vitro (such as the SMRT
RID
construct) fail to function in the mammalian two-hybrid assay; 4)
T3R mutants, such as v-Erb A, that exhibit a
hormone-resistant association with SMRT in vitro display a
hormone-resistant function in the two-hybrid assay; and 5) nuclear
hormone receptors that fail to interact with SMRT in vitro
(such as vitamin D3 receptor) fail to function in the
two-hybrid assay in vivo (Fig. 2A
).

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Figure 2. Tyrosine Kinase-Mediated Inhibition of the
Interaction between T3R and SMRT, Determined by a
Mammalian Two-Hybrid Assay
A, Mammalian cell-based two-hybrid assay of the interaction between
SMRT and T3R . A pSG5 vector expressing either a GAL4DBD
alone (empty DBD), a GAL4DBD fused with the receptor interaction domain
of SMRT (amino acids 751-1495; DBD-SMRT), or a GAL4DBD fused with a
subdomain of SMRT lacking the receptor interaction domain (amino acids
751-1074; DBD-SMRT RID), as indicated within each panel, were
introduced into CV-1 cells by calcium phosphate coprecipitation
together with a GAL4 (17-mer) luciferase reporter and a pSG5-GAL4-AD
construct. As indicated below the panels, the GAL4-AD
constructs contained either a GAL4-AD domain alone (empty), the
T3R hormone-binding domain (T3R), the same
T3R construct with a mutation that disrupts
SMRT-association (P156R), the analogous region of v-Erb
A (v-Erb A), or the vitamin D3 receptor hormone-binding
domain (VDR). The cells were subsequently incubated in the absence
(cross-hatched bars) or presence (solid
bars) of 1 µM cognate hormone, the cells were
harvested, and the relative luciferase activity was determined
normalized to that of a pCH110-lac Z plasmid introduced
as an internal control. The data represent the average and range of
duplicate experiments. B, V-Erb B inhibition of the
two-hybrid interaction between T3R and SMRT. A pSG5
vector expressing either an GAL4DBD alone (empty DBD), a GAL4DBD fused
with the receptor interaction domain of SMRT (amino acids 751-1495,
DBD-SMRT), or a GAL4DBD fused with the hormone- binding domain of
RXR were introduced into CV-1 cells by lipofection, together with
either the empty pSG5-GAL4-AD vector or the pSG5-GAL4-AD fused to the
hormone-binding domain of T3R . In addition, each transfection included
the GAL4 (17-mer) luciferase reporter, a pCMV-lac Z
internal standard, and the amount of the v-Erb B
expression plasmid indicated below the panel (0 to 200
ng). The cells were subsequently incubated and harvested, and the
luciferase activity was determined and normalized to ß-galactosidase
activity. The SMRT/T3R interactions were assayed in the absence of
T3.
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We next tested the effect of v-Erb B on this
SMRT/T3R interaction. The introduction of v-Erb
B virtually abolished the two-hybrid interaction between SMRT and
T3R, resulting in a greater than 20-fold inhibition of
luciferase expression (Fig. 2B
). This inhibition by v-Erb B
appeared to be operative on the SMRT/T3R interaction
itself: v-Erb B had little or no effect on the basal level
of reporter expression if either (or both) of the GAL4DBD-SMRT or
GAL4-AD-T3R fusions were omitted from the transfection
(Fig. 2B
). Similarly, v-Erb B had little or no effect on
expression of a ß-galactosidase reporter, lacking GAL4-binding sites,
employed in the same experiments as an internal control (data not
shown). We conclude that the effects of v-Erb B in the
two-hybrid assay require the presence of the T3R and SMRT
constructs and are therefore unlikely to be mediated by a nonspecific
inhibition of the activity of the reporter promoter itself, or by a
disruption of the stability or enzymatic activity of the encoded
luciferase protein.
We also wished to exclude the possibility that
v-Erb B was acting by inhibiting the expression or function
of either the GAL4-DBD or GAL4-AD moieties, rather than by interfering
with the SMRT/T3R interaction itself. We therefore assayed
the effects of v-Erb B on the two-hybrid interaction between
T3R and retinoid X receptor (RXR); RXRs are heterodimer
partners for T3Rs, and the two receptor classes can
physically associate in vitro and in vivo (6).
T3R exhibited a strong interaction with RXR in our
mammalian two-hybrid system that was relatively unaltered by
cointroduction of v-Erb B; only a very modest inhibition of
the T3R/RXR interaction was observed at high levels of
v-Erb B, perhaps reflecting very small differences in the
overall physiology of these cells in the presence or absence of the
tyrosine kinase. This relative lack of an effect of v-Erb B
on the T3R/RXR interaction was in notable contrast to the
dramatic v-Erb B-mediated inhibition of the
T3R/SMRT interaction (Fig. 2B
). These results indicate that
the interaction between SMRT and T3R is specifically
inhibited by cointroduction of an activated EGF-R/v-Erb B
allele.
We also repeated these experiments using the v-Erb A
oncoprotein derivative of T3R. In common with wild-type
T3R, the v-Erb A protein exhibited a strong
two-hybrid interaction with SMRT (Fig. 3
). This two-hybrid interaction was again
severely impaired by the cointroduction of v-Erb B. In
contrast, substitution of the pGAL4-AD-v-Erb A construct
with one containing a v-Erb A-mutant defective for SMRT
binding (v-Erb A P144R) abolished, as expected, both the
two-hybrid interaction and the effects of v-Erb B (Fig. 3
).
We conclude that v-Erb B signaling disrupts the ability of
SMRT to interact with both hormone-dependent and hormone-independent
forms of the T3R.

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Figure 3. Tyrosine Kinase Inhibition of the Mammalian
Two-Hybrid Interaction between SMRT and the v-Erb A
Derivative of T3R
The same protocol as in Fig. 2B was repeated, but utilizing either an
empty pSG5-GAL4-AD construct (empty), a
pSG5-GAL4-AD-v-Erb A fusion (v-Erb A), or
the same v-Erb A fusion bearing a mutation disrupting
the SMRT association domain (P144R). The transfections were performed
in the absence (-) or presence (+) of 100 ng of the
v-Erb B expression construct, as indicated
below the panel.
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Stimulation of Endogenous EGF-R Can Substitute for Transfection of
v-Erb B
The experiments described above relied on introduction of
v-Erb B as a means of strongly activating the EGF-R signal
transduction pathway. CV-1 cells express modest levels of endogenous
EGF-R, and we therefore extended our experiments to test the ability of
transforming growth factor-
(TGF-
), a physiological agonist for
EGF-R (52), to substitute for the transfection of v-Erb B.
Incubation of CV-1 cells with TGF resulted in a reproducible 2-fold
inhibition of the T3R/SMRT interaction (Fig. 4A
). A similar effect could be observed
with EGF (data not shown). Although the effects of TGF-
and EGF
incubation were much more modest than that observed with transfection
of v-Erb B, this differential is not unanticipated. When
transfected, v-Erb B is probably expressed at much higher
levels than is endogenous EGF-R and is likely to be a much stronger
inducer of downstream signal transduction than is EGF-R ligand. In
addition, v-Erb B is refractory to many of the negative
feedback mechanisms that constrain the actions of the wild-type EGF-R;
v-Erb B-induced signaling, therefore, is likely to be more
sustained than that of EGF or TGF ligand. We conclude that stimulation
of the EGF-R-signaling pathway, by either EGF-R itself or by a
ligand-independent form of EGF-R, results in inhibition of the
T3R/SMRT interaction, although the effect is more dramatic
with the latter.

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Figure 4. Inhibition of the Mammalian Two-Hybrid Interaction
between SMRT and v-Erb A by Various Signal
Effectors/Transducers
The same protocol as in Fig. 2B was repeated, but testing the ability
of different signal effectors/transducers to replace
v-Erb B. A, Effect of transforming growth factor
(TGF) on the mammalian two-hybrid interaction between SMRT and
v-Erb A. pSG5 vectors expressing the GAL4-DBD-SMRT
fusion were introduced into CV-1 cells together with the GAL4 (17-mer)
luciferase reporter and either an empty pSG5-GAL4-AD or the
pSG5-GAL4-AD-v-Erb A fusion, as indicated
below the panel. The cells were then treated
(solid bars) or not (cross-hatched bars)
with 50 µM TGF and harvested, and the relative
luciferase activity, normalized to ß-galactosidase activity, was
determined. The data represent the average and range of duplicate
experiments. B, Effect of different signal transducers on the mammalian
two-hybrid interaction between SMRT and v-Erb A. The
CV-1 cells were transfected as described in panel A, and either not
manipulated further (None), treated with the concentrations of
8-bromo-cAMP indicated (cyclic AMP), or transfected with expression
plasmids for v-Erb B, v-Ras,
v-Raf, or the p110 catalytic subunit of
phosphatidylinositol-3-kinase (PI3-K), as indicated
below the panel. The cells were subsequently harvested,
and the relative luciferase activity, normalized to ß-galactosidase
activity, was determined. The data represent the average and range of
duplicate experiments.
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We also performed a preliminary dissection of the downstream signaling
pathways that may be operating in this EGF-R/v-Erb B
signaling pathway. Ras has been implicated as one (of
several) downstream effectors involved in EGF-R signaling (48):
notably, substitution of v-Erb B with v-Ras also
inhibited the two-hybrid interaction between T3R and SMRT,
although not as strongly as did v-Erb B itself (Fig. 4B
).
cAMP also exerted a modest inhibition, but only at relatively high
concentrations that also lead to detectable cell toxicity (Fig. 4B
). In
contrast, introduction of a number of other potential signal
transducers, such as v-Raf and the catalytic subunit of
phosphatidyl-inositol 3-kinase, failed to inhibit the
T3R/SMRT interaction (Fig. 4B
). We conclude that
c-Ras may be one downstream mediator of the v-Erb
B effects on corepressor function, but that other pathways are also
likely to contribute to the effects observed here.
Activation of the EGF-R Signaling Pathway Inhibits the Ability of
SMRT to Interact with RARs, and with the PLZF Transcriptional
Repressor
RARs share the ability to bind to the SMRT corepressor complex and
to repress target gene transcription in vivo. Nonetheless,
RARs diverge from T3Rs in primary amino acid sequence,
hormone specificity, and target gene specificity (7). We therefore
tested the effects of the EGF-R signaling pathway on the SMRT/RAR
interaction. In the absence of v-Erb B, a strong two-hybrid
interaction was observed between RAR
and SMRT that could be
inhibited, as expected, by cognate hormone (Fig. 5
). Introduction of increasing amounts of
v-Erb B virtually abolished this interaction, resulting in a
more than 10-fold reduction in GAL4(17-mer) luciferase reporter
activity (Fig. 5
). As a negative control we examined the effects of
v-Erb B on the two-hybrid interaction between RAR and RXR
[RXRs are heterodimer partners for RARs as well as for
T3Rs (6)]; consistent with the results we observed for
T3R, the two-hybrid interaction between RAR and RXR was
relatively unaffected, or even slightly stimulated, by coexpression of
v-Erb B (Fig. 5
).
In addition to nuclear hormone receptors, SMRT has also been implicated
in transcriptional repression mediated by many nonreceptor
transcription factors, such as the PLZF protein (26, 27, 28). PLZF
possesses little or no detectable amino acid relatedness to the nuclear
hormone receptors, and the interactions between PLZF and SMRT appear to
be mediated by protein determinants distinct from those implicated in
the nuclear hormone receptor interaction (26). Once again, however, the
two-hybrid interaction between PLZF and SMRT observed in CV-1 cells in
the absence of v-Erb B was strongly inhibited in the
presence of v-Erb B (Fig. 6B
).
Consistent with this v-Erb B-mediated inhibition of the
interaction between PLZF and SMRT, cointroduction of v-Erb B
counteracted PLZF-mediated transcriptional repression when tested using
a suitable PLZF-fusion/reporter construct system (Fig. 6A
). We conclude
that the effects of tyrosine kinase signaling on SMRT function are
manifested on a variety of transcriptional repressors.

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Figure 6. Tyrosine Kinase-Mediated Inhibition of
Transcriptional Repression by PLZF and of the Two-Hybrid Interaction
between PLZF and SMRT
A, Inhibition by v-Erb B of transcriptional repression
by PLZF. The GAL4 (17-mer) luciferase reporter was introduced by
lipofection into CV-1 cells, together with the concentrations of a
pSG5-GAL4-DBD fusion of PLZF (amino acids 1456) indicated
below the panel. The transfections were performed in the
absence (cross-hatched bars) or presence (solid
bars) of a v-Erb B expression plasmid; the cells
were incubated and harvested, and the luciferase activity was
determined relative to that of the pCMV-lac Z reporter
employed as an internal transfection standard. Fold repression was
calculated relative to the levels of reporter gene expression detected
in the absence of the PLZF plasmid. B, Tyrosine kinase inhibition of
the mammalian two-hybrid interaction between SMRT and PLZF. The same
protocol as in Fig. 2B was repeated, but utilizing either an empty
pSG5-GAL4-AD plasmid ("empty") or a pSG5-GAL4-AD-PLZF fusion
("v-Erb A"), together with the pSG5-GAL4DBD-SMRT
construct. The transfections were performed in the absence
(cross-hatched bars) or presence (solid
bars) of the v-Erb B expression plasmid.
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There are two distinct domains of SMRT that mediate the interaction of
corepressor with T3R and with v-Erb A in
vitro and in vivo; these are denoted SMRT receptor
interaction domains (RID)-1 and RID-2 (12, 14, 15, 16). We therefore next
tested whether the inhibitory effects of v-Erb B were
specific to one or the other SMRT domain. In the absence of
v-Erb B, both RID-1 (SMRT amino acids 10551291) and RID-2
(SMRT amino acids 12911495) independently conferred a two-hybrid
interaction with v-Erb A, with the RID-2/v-Erb A
interaction somewhat the stronger of the two (Fig. 7A
). No interaction was observed with a
region of SMRT mapping upstream of the RID domains (SMRT amino acids
751-1074). Notably, both the RID-1 and RID-2 interactions with
v-Erb A were clearly inhibited by cointroduction of
v-Erb B, although the magnitude of this inhibition was
somewhat greater for the RID-1 interaction. In contrast to the ability
of v-Erb A and T3Rs to contact both RID-1 and
RID-2 regions of SMRT, RARs contact primarily the RID-1 domain (SMRT
amino acids 10551291; Ref. 14). Consistent with our results for
v-Erb A, the two-hybrid interaction between RAR and the SMRT
RID-1 domain was also strongly inhibited by the introduction of
v-Erb B (Fig. 7B
). We conclude that tyrosine kinase
signaling exerts an overall inhibition of the interaction between SMRT
and nuclear hormone receptors, with this effect particularly pronounced
for the interactions manifested by the SMRT RID-1 domain.
The Tyrosine Kinase-Mediated Inhibition of the RAR/SMRT Interaction
Observed in Vivo Is Also Observed in Vitro
We next exploited a protein-protein binding assay to determine
whether the tyrosine-kinase signaling effects in vivo could
be mimicked in an in vitro system. In this assay,
radiolabeled RAR
, synthesized by in vitro transcription
and translation, was tested for the ability to bind to an immobilized
glutathione-S-transferase (GST)-SMRT construct, synthesized
in and purified from Escherichia coli. RAR
bound to
the GST-SMRT was subsequently eluted and analyzed by SDS-PAGE. As
previously shown, RAR strongly bound to the GST-SMRT fusion under these
conditions, but not to nonrecombinant GST (Fig. 8
). Addition of lysates of control COS-1
cells, transfected with an empty pSG5 plasmid, had little or no effect
on the GST-SMRT/RAR interaction. In contrast, addition of lysates of
COS-1 cells transfected by v-Erb B inhibited the ability of
radiolabeled RAR to bind to the GST-SMRT matrix in vitro
(Fig. 8
). We conclude that activation of the EGF-R/v-Erb B
signaling pathway in CV-1 cells results in the production of an
inhibitory activity that can subsequently function to interfere with
the physical association between RAR and SMRT in vitro.
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DISCUSSION
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Transcriptional Repression by Nuclear Hormone Receptors Is Opposed
by Tyrosine Kinase-Mediated Signaling
Although originally believed to be regulated exclusively by
cognate hormone, many nuclear hormone receptors are actually subject to
control by an assortment of nonligand signals. For example, the nature
of the DNA-binding site, dimerization with other nuclear hormone
receptors, protein-protein interactions with other nonreceptor
proteins, and covalent modifications, such as receptor phosphorylation,
can all alter nuclear hormone receptor function (11, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47). These
extrahormonal signals can exert both positive and negative effects on
receptor activity in the absence of hormone and can either enhance or
preclude the effects of hormone. In this manuscript, we report that
tyrosine kinase signaling, such as that mediated by
EGF-R/v-Erb B, can specifically abrogate transcriptional
repression by the T3R, with little or no effect on
transcriptional activation. Thus, the transcriptional properties of
T3R are subject to regulation both by T3
hormone and by a distinct set of signals arising from tyrosine
kinase/growth factor receptors.
What is the nature of this tyrosine kinase-initiated inhibition of
T3R-mediated repression? As demonstrated here, activation
of the EGF-R/v-Erb B signaling pathway virtually abolished
the ability of SMRT to interact with T3R in a mammalian
two-hybrid assay. Although the two-hybrid assay is inherently an
indirect measure of protein-protein interaction, our experiments
strongly indicate that the effect of tyrosine kinase signaling is to
inhibit the actual interaction between T3R and SMRT
corepressor, rather than to interfere with an incidental or extraneous
aspect of the two-hybrid assay itself. For example, tyrosine kinase
signaling had little or no effect on the two-hybrid interaction between
T3R
and its heterodimer partner RXR (or between RAR
and RXR) in an assay dependent on virtually the same reagents and
subject to the same constraints as the T3R/SMRT two-hybrid
assay. Additional controls demonstrated that EGF-R/v-Erb B
had little or no effect on basal reporter gene function, on the
enzymatic activity of the luciferase itself, or on the DNA binding or
transactivation properties of the GAL4DBD or GAL4-AD fusions. Notably,
the inhibitory effects of EGF-R/v-Erb B signaling are also
demonstrable in vitro, at a reduced intensity, using
extracts of transfected cells in a protein-protein binding protocol.
These results further support our hypothesis that the tyrosine
kinase-signaling pathways act to disrupt the physical interaction
between unliganded nuclear hormone receptor and the SMRT
corepressor.
The Inhibitory Effects of Tyrosine Kinase Signaling Extend to SMRT
Interactions with v-Erb A, with RARs, and with PLZF
The SMRT corepressor complex has been implicated in
transcriptional silencing by T3Rs, by RARs, and by an
assortment of nonreceptor transcriptional repressors, such as BCL-6 and
PLZF (9, 13, 14, 15, 16, 17, 18, 20, 25, 26, 27, 28). Paralleling the inhibition of the
SMRT/T3R interaction, the capacity of SMRT to interact with
RARs and with PLZF was also dramatically inhibited by activation of the
EGF-R/v-Erb B-signaling pathway. Thus, the SMRT corepressor
may represent a common regulatory link through which gene silencing by
an assortment of transcription factors can be controlled by tyrosine
kinase signaling.
Although RARs and T3Rs possess distinct hormone and DNA
recognition properties, they share clusters of related amino acid
sequence and interact with overlapping, if somewhat distinct, portions
of SMRT. In contrast, PLZF possesses no detectable amino acid
relatedness with the nuclear hormone receptors and appears to interact
with SMRT through a POZ protein motif not present in the nuclear
hormone receptors (26, 27, 28). We do not yet know the precise nature of
the tyrosine kinase-initiated inhibitory effect, although our results
imply that whatever the molecular basis of this phenomenon, the
components and mechanism must be able to survive cell lysis to function
in vitro. Given the near-identical effects of tyrosine
kinase signaling on T3R-, RAR-, and PLZF-mediated
silencing, it is tempting to conclude that it is the shared SMRT
corepressor, not these divergent transcription factors, that represents
the actual target of this inhibition. Plausible mechanisms may
therefore include the covalent modification of SMRT or of another
component of the corepressor complex or, alternatively, the altered
expression or altered function of a modulator of SMRT function. Of
course, we cannot fully exclude that the transcription factors are
themselves the targets of the actions of v-Erb B, possibly
through the actions of an as-yet-unidentified, but shared
modulator.
Our preliminary use of known components of the signal transduction
pathway that lie downstream of EGF-R/v-Erb B suggests that
the effects of v-Erb B on SMRT function can be partially
mimicked by v-Ras, but not by v-Raf, protein
kinase C, or moderate levels of cAMP. It is somewhat intriguing that
v-Raf does not mimic the actions of v-Ras in the
inhibition of SMRT function; presumably the actions of v-Ras
in this context are not mediated through c-Raf, or perhaps
v-Raf lacks some functions present in the c-Raf
protein. It is also important to note that v-Ras appears
capable of only partial substitution for v-Erb B in this
phenotype; further work is in progress to better define the signal
transduction pathway involved and to identify any additional
intermediates and effectors.
Tyrosine Kinases and Transcriptional Repression: A Consistent
Theme
A multiplicity of circumstantial linkages have been documented
between the actions of the nuclear hormone receptors and the signal
transduction events initiated by tyrosine kinases. For example, as
already described, the two oncogenes encoded by the avian
erythroblastosis virus, v-erb A and v-erb B, are
aberrant versions of the normal cellular genes for T3Rs and
EGF-R, and these two oncogenes function synergistically in
erythroleukemogenesis. V-Erb A has been proposed to function
in oncogenesis as a transcriptional repressor (49, 50, 51). This may appear
at first to be contradictory to our own observations that
v-Erb B counteracts repression by v-Erb A in CV-1
cells. However, the precise actions of v-Erb A in the
erythroleukemic cell remain incompletely understood and may extend
beyond simple transcriptional repression, perhaps involving functions
that are enhanced by v-Erb B. Alternatively, the
v-Erb B signal transduction pathway in avian erythroleukemic
cells may be different from that in the primate-derived CV-1 kidney
cells employed here and may not result in an antagonism of
v-Erb A repression in the former.
A variety of other tantalizing links have been established between the
actions of tyrosine kinases and those of nuclear hormone receptors. EGF
treatment, for example, has been reported to activate the estrogen
receptor, even in the absence of estrogen agonist, possibly through
phosphorylation of mitogen-activated protein kinase sites in
the nuclear receptor and/or by phosphorylation of tyrosine 537 in its
ligand-binding domain (e.g. Refs. 32, 38, 41, 53, 54).
This observation is likely to have important medical implications:
mammary carcinomas that display aberrant estrogen receptor function
often also exhibit aberrant expression of EGF-R family members, such as
the closely related HER-2 tyrosine kinase (55, 56). More broadly,
interactions between tyrosine kinases and transcriptional repressors
occur in a variety of eukaryotic systems. In Drosophila
embryonic development, for example, activation of torso, a
membrane-associated tyrosine kinase, inhibits transcriptional
repression by dorsal, an ortholog of mammalian NF-
B (57).
These interactions between tyrosine kinases and nuclear hormone
receptors can operate in both directions: for example, the promoter for
human EGF-R contains a binding site for T3R, and endogenous
EGF-R expression is repressed by T3R in at least certain
cell types (58).
Notably, the SMRT/N-CoR corepressor has also been implicated in
mediating the inhibitory effects of certain ligand antagonists on the
progesterone and estrogen receptors (47, 59, 60). When this manuscript
was in the final stages of preparation, McDonnell and colleagues (47)
reported that cAMP treatment can counteract the effect of antagonists
on progesterone receptor, apparently by interfering with corepressor
function (47). Although we observe a much more modest effect of cAMP on
the receptors and transcription factors tested here, it appears
reasonable that the progesterone receptor studies, and our own, reflect
a common phenomenon by which transcriptional repression is made
subservient to a variety of cellular signal transduction pathways.
These observations may also prove relevant to the actions of chicken
ovalbumin upstream promoter transcription factor (COUP-TF), a
member of the orphan class of nuclear hormone receptors (61). COUP-TF
lacks a known hormone ligand and functions in most contexts as a strong
transcriptional repressor, apparently through the ability of COUP-TF to
bind to the SMRT/N-CoR corepressor complex (61). Intriguingly, COUP-TF
repression is strongly counteracted by dopamine, by cAMP, and by
okadaic acid, even though none of these reagents bind directly to the
COUP-TF protein (62). Given the results presented here and by McDonnell
and co-workers, it will be very interesting to determine whether the
actions of these nonligand signals in COUP-TF-mediated regulation are
mediated through a similar signal transduction pathway that leads to
disruption of the ability of SMRT to interact with COUP-TF.
In conclusion, we have elucidated a previously unrecognized mechanism
through which the functions of the nuclear hormone receptors (and of
nonreceptor transcription factors such as PLZF) can be regulated. This
mechanism involves tyrosine kinase signal transduction pathways that
alter the interactions of these transcription factors with essential
cofactors, such as the SMRT corepressor complex. These alterations to
corepressor function are likely to represent an important component of
signal convergence and integration in eukaryotic gene regulation.
 |
MATERIALS AND METHODS
|
---|
Transient Transfections
Transfections of CV-1 cells were performed by a
lipofectin-mediated method (19). Approximately 4 x
105 cells were transfected with 50 ng of pSG5-T3R
or
pSG5-v-erb A plasmid, 100 ng of pCMV-lac Z as an
internal control, and 100 ng of ptk-luc-TRE with or without 200 ng of a
pSG5-v-erb B expression plasmid. Cells were harvested at
48 h post transfection. Relative luciferase activity was measured
and normalized to ß-galactosidase activity (19).
Mammalian Two-Hybrid Assays
Construction of the pSG5 GAL4 DNA DBD-SMRT derivatives and
GAL4-AD-T3R
vectors were previously described (26). The
pSG5 GAL4-AD-RAR
and RXR
vectors were constructed by inserting
the appropriate PCR-generated DNA fragments, bearing terminal
EcoRV and XhoI sites, into the pSG5 GAL4-AD
vector (26). If a calcium phosphate coprecipitation protocol was
employed, the transfections were performed in 60-mm plates containing
approximately 2.5 x 105 CV-1 cells. Each plate was
transfected with 500 ng each of the pSG5 GAL4DBD vector, the pSG5
GAL4-AD vector, the pGL2-GAL4(17-mer)-luciferase reporter, and a
pCH110-lac Z vector (employed as an internal control). If
the lipofectin protocol was employed, the transfections were performed
in 12-well culture plates, each well containing 7 x
104 CV-1 cells; each well was transfected with 25 ng of the
pSG5 GAL4DBD vector, 100 ng of the pSG5 GAL4-AD vector, 100 ng of
pGL2-GAL4(17-mer)-luciferase reporter, and 100 ng of a
pCMV-lac Z as an internal control, with either 200 ng of an
empty pSG5 vector or 200 ng of the pSG5 v-erb B plasmid.
Luciferase and ß-galactosidase assays were performed as previously
described (19).
In Vitro Receptor/Corepressor Binding Assays
The nonrecombinant GST and the GST-SMRT (codons 10551291)
constructions were created in a pGEX-KG vector background (14, 18, 19, 26). GST-fusion proteins were expressed in E. coli, were
purified, and were immobilized on glutathione agarose as previously
described (14, 18, 19, 26). The immobilized GST fusion proteins were
then incubated at 37 C for 30 min with lysates of COS-1 cells, prepared
as described below, and then incubated for 1 h at 4 C with
35S-labeled RAR
, prepared in a coupled in
vitro transcription and translation system (Promega TnT procedure,
Promega, Madison, WI). The agarose matrix was extensively washed, and
the bound proteins were eluted with free glutathione and were analyzed
by SDS-PAGE (18, 19, 26). The electrophoretograms were visualized by
autoradiography and were quantified by PhosphorImager analysis
(Molecular Dynamics STORM system, Molecular Dynamics, Sunnyvale,
CA).
Cell extracts were prepared as follows: COS-1 cells, maintained in
60-mm tissue culture dishes, were transiently transfected with 1 µg
per dish of either an empty pSG5 vector or a pSG5 v-Erb B
expression vector, using the lipofectin-mediated method. At 60
h post transfection, the cells were harvested, and then lysed by
sonication in WCE buffer (25 mM HEPES, pH 7.7, 0.3
M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA,
0.1% Triton X-100, 0.5 mM dithiothreitol, 20
µM ß-glycerolphosphate, 0.1 mM
Na3VO4, 2 µg/ml leupeptin, and 100 µg/ml
phenylmethylsulfonyl fluoride).
 |
ACKNOWLEDGMENTS
|
---|
We thank H.-J. Kung, B. Vennstrom, and A. Dejean for generously
providing molecular clones employed in this research.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Martin L. Privalsky, Section of Microbiology, Division of Biological Sciences, University of California at Davis, 1 Shields Avenue, Davis, California 95616. E-mail:
mlprivalsky{at}ucdavis.edu
This work was supported by Public Health Service/NIH Grants R37
CA-53394 and R01 DK-53528.
Received for publication March 30, 1998.
Revision received May 6, 1998.
Accepted for publication May 12, 1998.
 |
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