From the Section of Microbiology and Section of Molecular and
Cellular Biology, Division of Biological Sciences, University of
California, Davis, California 95616
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INTRODUCTION |
The nuclear hormone receptors are a family of interrelated
proteins that regulate the transcription of specific target genes in
response to binding of cognate hormone ligand (1-8). They include the
steroid hormone receptors, retinoic acid receptors, vitamin D3
receptor, and the thyroid hormone receptors
(T3R
1 and T3R
). The
nuclear hormone receptors play many critical roles in vertebrate
homeostasis, morphogenesis, development, and reproduction; in addition,
aberrant nuclear hormone receptors have been implicated as causal
agents in oncogenesis and in endocrine disorders (1-8). For example,
the v-ERB A oncoprotein is a neoplastic retroviral derivative of T3R
(9, 10). T3Rs generally repress transcription in the absence of hormone
and activate transcription in the presence of hormone (1-8). Although
retaining the ability to bind to DNA, v-ERB A has sustained multiple
mutations relative to the T3R
progenitor and has lost the ability to
activate transcription directly (Fig. 1
and Refs. 11-13). Instead, v-ERB A functions in most contexts as a
constitutive repressor and can interfere with target gene activation by
a variety of nuclear hormone receptors, including T3Rs, retinoic acid
receptors, and estrogen receptors (11-17). Therefore, v-ERB A is
viewed as a dominant negative oncoprotein that acts in the cancer cell
by antagonizing the actions of normal cellular receptors.

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Fig. 1.
Schematic of avian T3R -1 and v-ERB A
proteins. A schematic representation of the protein structure of
avian T3R -1 (top) and v-ERB A (bottom) are
illustrated (A). Regions within the T3R structure implicated
in DNA recognition (including a zinc finger motif, Zn motif) and in
hormone binding are indicated. Vertical bars in the v-ERB A
diagram indicate the location of amino acid substitutions relative to
the avian T3R -1 sequence; gag refers to retroviral
structural sequences present on the N terminus of the viral protein.
The location of the serine residues (serines 28 and 29) that are
targets for PKA phosphorylation are indicated in the avian T3R -1
sequence; the analogous serines 16 and 17 are also shown in the v-ERB A
schematic. An amino acid sequence alignment is also presented
(B) comparing the N-terminal domain of avian T3R -1
(top) with that of v-ERB A (bottom), with the
potential sites of PKA phosphorylation represented in
boldface.
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Nuclear hormone receptors recognize their target genes by binding to
DNA sequences referred to as hormone response elements or HREs (1-8).
Although DNA binding by these receptors is principally mediated by a
"zinc finger" motif near the center of each receptor (Fig.
1A), additional flanking domains, both N- and C-terminal of
the zinc finger itself, contribute to the DNA recognition specificity of each receptor (18-26). Most nuclear hormone receptors can bind to
DNA as protein dimers, with each receptor molecule recognizing a
"half-site," a conserved 6-8-base DNA sequence (1-8, 27, 28).
HREs have therefore been traditionally viewed as composed of two
half-sites, with the sequence, spacing, and orientation of the
individual half-sites contributing to the specificity of DNA
recognition (1-8). However, recent work has demonstrated that receptor
dimers are not the only paradigm for DNA recognition and that certain
members of the nuclear receptor family can also bind to single DNA
half-sites as protein monomers or can bind to highly reiterated DNA
half-sites as receptor oligomers larger than dimer in size
(29-35).
Nuclear hormone receptors are substrates for a variety of protein
kinases, and phosphorylation can have profound effects on the
subcellular localization, DNA binding, and transcriptional activity of
these receptors (36-40). For example, both v-ERB A, and its
progenitor, avian T3R
-1, are phosphorylated by protein kinase A
(PKA) in vitro and, apparently, in vivo (41, 42). The major site of PKA phosphorylation in v-ERB A has been mapped to
serine 16/serine 17 (Fig. 1A); both amino acids represent
consensus PKA sites, and prior studies did not determine which of the
two, or if both, were phosphorylated (42). The same PKA site(s) also exist and are phosphorylated in the avian T3R
-1 progenitor (denoted serine 28/serine 29 in the T3R-numbering system; Fig. 1A).
Significantly, preventing the phosphorylation of serine 16/17 in v-ERB
A, either by substitution of alanines or by use of kinase inhibitors,
dramatically impairs oncogenic transformation (41). In contrast,
conversion of serines 16/17 to threonines (which retain the ability to
be phosphorylated by PKA) preserves v-ERB A oncogenic activity (41). Alterations in PKA activity have also been reported to modify T3R
-1
activity in cells (38, 43).
Although it appears that the phosphorylation of the PKA sites of v-ERB
A (and probably of avian T3R
-1) is critical for full function, the
molecular mechanism behind this phenomenon has not been established.
The PKA phosphorylation sites in v-ERB A/T3R
-1 are within a domain
that we have previously implicated as contributing to DNA sequence
recognition by these receptors, suggesting that phosphorylation could
potentially affect the DNA binding properties of these proteins (22,
23, 25, 26, 42). In this paper we show that phosphorylation of either
v-ERB A or T3R
-1 has little or no effect on the overall affinity of
receptor for DNA. However, phosphorylation does nonetheless influence
DNA recognition by severely inhibiting the ability of these receptors
to bind to DNA as protein monomers, without significantly affecting
dimer binding. These studies suggest that the phosphorylation of v-ERB A and T3R
-1 by PKA may regulate whether these proteins will bind to
DNA elements containing monomeric half-sites versus dimeric repeats and may thereby provide a means of regulating promoter recognition in vivo.
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EXPERIMENTAL PROCEDURES |
Mutagenesis of T3R
-1 and v-ERB A Phosphorylation
Sites--
All mutants were created by a two-step polymerase chain
reaction mutagenesis technique (44). The wild-type avian T3R
-1 clone
was used as the initial template for mutagenesis (9). The flanking
primers, which hybridize to sequences in the parental pGEX2T vector,
were 5'-GAATG GAATT CTCAT GGAAC AGAAG CCCAG C-3' and 5'-AATAA GGAAT
TCCCT ACACC TCCTG GTCCT-3'. The internal oligonucleotide primers used
to create the S28A mutants were 5'-AGAAA GGCCA GCCAA TGTTT GGTGA A-3'
and 5'-TGGCT GGCCT TTCTT TTGCG CTTGC C-3'. The internal oligonucleotide
primers used to create the S29A mutants were 5'-AAGAG CGCCC AATGT TTGGT
GAAGA G-3' and 5'-CATTG GGCGC TCTTT CTTTT GCGCT T-3'. The double mutant
(S28A/S29A) of the avian T3R
-1 was created using DNA from the S28A
mutant as a template and with 5'-AGAAA GGCCG CCCAA TGTTT GGTGA A-3' and
5'-TGGGC GGCCT TTCTT TTGCG CTTGC C-3' as the internal oligonucleotide
primers. The N-terminal deletion (
N-T3R) of avian T3R
-1, which
deletes codons 3-45, was described previously (25). All mutations were subsequently confirmed by sequence analysis.
Molecular Cloning and Preparation of Recombinant
Proteins--
The wild type T3R
-1, (S28A)T3R
-1, (S29A)T3R
-1,
(S28/29A)T3R
-1,
N-T3R
-1, and wild type v-ERB A
sequences were cloned as EcoRI to EcoRI fragments
into the baculovirus transfer vector pVL1393 (17). Appropriate
baculovirus clones, expressing the protein of interest, were obtained
by in vivo recombination and plaque purification and were
used to infect Sf9 cells to generate nuclear preparations
containing wild-type or mutant T3R/v-ERB A proteins (17, 22, 26).
Alternatively, the wild type, single point mutants, and double mutants
of T3R
-1 and v-ERB A were also cloned as EcoRI
to EcoRI fragments into pGEX-2T, and the resulting glutathione S-transferase (GST) fusion proteins were
isolated from transformed Escherichia coli as described
previously (45).
Preparation of Oligonucleotide Probes for the DNA Binding
Assay--
Three different DNA sequences were used as DNA binding
probes as follows: a direct repeat element with a 4-base spacer (DR-4, 5'-TCGAC TCAGG TCACA GGAGG TCAGA G-3') a
inverted repeat element (TREpal, 5'-TCGAG ATCTC AGGTC ATGAC
CTGAG ATC-3'), and an element containing a single half-site (1S,
5'-TCGAC TGAGG TCACT ACATA G-3'). Each probe was
synthesized as two complementary oligonucleotides with 4-base
overhangs; the complementary sequences were annealed, and the
double-stranded DNAs were radiolabeled by extension using [
-32P]dGTP and the Klenow fragment of DNA
polymerase.
Electrophoretic Mobility Shift Assays--
Wild-type or mutant
receptors (estimated as 1 ng per reaction, unless otherwise noted) were
incubated with the purified catalytic subunit of PKA (either purchased
from Promega or the gift of Dr. D. Walsh, University of California,
Davis) in 6 µl of protein kinase buffer (40 mM Tris-HCl,
pH 7.4, 20 mM magnesium acetate) in the presence or absence
of rATP (200 µM) for 20 min at 30 °C. Non-recombinant
GST or baculovirus extracts were employed in parallel as negative
controls. Binding buffer (10 mM Tris-HCl, pH 7.5, 3%
glycerol, 13.3 µg/µl bovine serum albumin, 66.7 mM KCl,
2 mM MgCl2, and 133 mg/ml poly(deoxyinosinic
acid-deoxycytidylic acid)) and radiolabeled oligonucleotide probe
(25,000-40,000 cpm, 20-60 ng of DNA) were then added to each sample
to a final volume of 15 µl, and the samples were incubated for 25 min
at room temperature. Samples were then resolved by electrophoresis
through a 5% polyacrylamide gel at 200 V for 75 min at room
temperature (25, 26, 33). The gels were then dried and analyzed using
the Storm PhosphorImaging system and ImageQuaNT software (Molecular
Dynamics).
In Vitro Phosphorylation Assay--
Receptors were incubated
with PKA for 20 min at 30 °C in a total volume of 20 µl of protein
kinase buffer containing 1 µCi of [
-32P]rATP at a
final specific activity of 18 Ci/mmol (NEN Life Science Products).
After incubation, the phosphorylated proteins were analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed
by autoradiography and/or PhosphorImager analysis.
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RESULTS |
Phosphorylation of v-ERB A and T3R
-1 Alters the Proportion of
Receptor Monomer to Receptor Dimer Complexes Formed on DNA--
We
first wished to establish that the recombinant avian T3R
-1 and v-ERB
A proteins synthesized in a baculovirus/Sf9 cell system could be
phosphorylated in vitro by PKA. The wild-type T3R
protein, the v-ERB A protein, or equivalent nuclear extracts of
Sf9 cells infected by a non-recombinant baculovirus were
incubated with purified PKA and analyzed by SDS-PAGE and
autoradiography (Fig. 2). The T3R
and
v-ERB A proteins, detected by Coomassie Blue stain (Fig.
2A), were both efficiently radiolabeled by this procedure
(Fig. 2B), whereas no equivalent 32P-labeled
bands were detected in the non-recombinant extracts (Fig.
2B) or in the absence of added PKA (data not shown). The amount of 32P radiolabel incorporated was proportional to
the PKA concentration up to a maximum stoichiometry, calculated from
specific activity criteria, of approximately 2.2 (± 0.2) mol of
phosphate per mol of receptor. Similarly, PKA was able to radiolabel
purified GST fusions of T3R
-1 and v-ERB A but not non-recombinant
GST (data not shown).

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Fig. 2.
Avian T3R -1 and v-ERB A are phosphorylated
by PKA in vitro. Extracts of Sf9 cells infected by
baculovirus expressing avian T3R -1 (T3R), v-ERB A
(v-ERB A), or by non-recombinant baculovirus
(None) were prepared as described in the text. Aliquots of
these extracts were either directly resolved by SDS-PAGE and visualized
by Coomassie Blue staining (A) or were first incubated with
PKA (0.72 units per reaction) and [ -32P]rATP, then
resolved by SDS-PAGE, and visualized by autoradiography (B).
Alternatively, the extracts were employed in an electrophoretic gel
shift assay using a radiolabeled DR-4 DNA element as a probe
(C); the position of receptor monomer (1R) and
receptor dimer (2R) complexes bound to the DNA are
indicated, as is the position of free (unbound) probe.
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We next used electrophoretic mobility shift assays to determine if PKA
phosphorylation altered the DNA binding properties of T3R
and v-ERB
A. We first tested a DNA probe containing a prototypic HRE composed of
a direct repeat of two AGGTCA half-sites, separated by 4 base pairs
(denoted a DR-4). Untreated T3R
or v-ERB A both efficiently bound to
the DR-4 element, each forming two distinct protein-DNA complexes
(denoted 1R and 2R in Fig. 2C); this
property has been widely noted previously and reflects the ability of
the T3R and v-ERB A proteins to bind to DNA either as protein monomers
(the faster migrating 1R complexes) or, alternatively, as protein
"dimers" (the slower migrating 2R complexes) (e.g. Refs.
29-32, 43, 46, and 47). This interpretation of the electrophoretic
mobility shift is fully supported by our own data as follows.
(a) Only the faster migrating monomer complex was detected
when using a single half-site element as the DNA probe, whereas
response elements containing two half-sites in a variety of
orientations were bound as a mix of monomer and dimer complexes (Fig.
3). (b) Thyroid hormone
selectively destabilized the slower migrating species (data not shown),
a property specific for the homodimeric form of T3R (29-32, 46, 47).
(c) RXRs form heterodimers with T3Rs, and addition of RXR to
our binding reactions converted both monomer and homodimer T3R species
to a distinct complex migrating at a position characteristic of
heterodimers (Fig. 5).

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Fig. 3.
Phosphorylation by PKA inhibits DNA binding
by T3R and v-ERB A monomers. Either a T3R preparation
(A) or a v-ERB A preparation (B), isolated from
baculovirus-infected Sf9 cells, was incubated with 0.72 units of
PKA (lanes 1 and 4; yielding an estimated 0.9 mol
of phosphate per mol of receptor), 7.2 units of PKA (lanes 2 and 5; yielding an estimated 2 mol of phosphate per mol of
receptor), or without enzyme (lanes 3 and 6) in
the presence or the absence of rATP. The receptor preparations were
then employed in an electrophoretic gel shift assay using a DR-4 DNA
element as the radiolabeled probe and visualized by autoradiography.
Alternatively, receptor preparations were treated and employed in an
electrophoretic shift assay in an analogous fashion but using a TREpal
element (C) or a single half-site element (D) as
the radiolabeled DNA probe. All incubations in C and
D were performed in the presence of rATP. 1R and
2R indicate the positions of receptor monomer and dimer
complexes respectively; Free Probe indicates the position of
unbound DNA.
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Significantly, although untreated (or mock-treated) T3R
and v-ERB A
proteins bound to the DR-4 element as a mixture of monomers and dimers,
PKA treatment of otherwise identical preparations of T3R
or v-ERB A
leads to the almost exclusive formation of dimeric complexes (Fig. 3,
A and B; compare the PKA-treated receptor preparations, lanes 1 and 2, to the mock-treated
receptor preparations, lane 3). This effect was proportional
to PKA concentration, was observed with a variety of purified PKA
preparations, and was not detected if rATP was omitted from the kinase
buffer, suggesting that it is indeed the phosphorylation of the
receptors that is responsible for this phenomenon (Fig. 3, A
and B, compare lanes 4-6 with lanes
1-3; and data not shown). Similarly, binding of T3R
and v-ERB
A to a palindromic DNA element (TREpal, Fig. 3C) also showed a reproducible decrease in monomer binding on PKA treatment
(although the reduction on the palindromic element was as not as
dramatic as that observed with the DR-4 element). We conclude that PKA
phosphorylation of either T3R
-1 or v-ERB A significantly alters the
ratio of receptors binding to DNA as protein monomers versus
dimers.
Phosphorylation by PKA appears to specifically destabilize the ability
of receptor monomers to bind DNA, rather than alter the absolute
affinity of the receptor for DNA or modify the dimerization properties
of the phosphorylated receptor. Several possible mechanisms could
account for the observed reduction of monomer binding by PKA treatment
of T3R
and v-ERB A. Phosphorylation could enhance the
protein-protein interaction between receptor molecules, resulting in an
increased cooperativity on binding DNA. Alternatively, phosphorylation might conceivably increase the overall affinity of receptor for DNA. In
either of these scenarios, the effect of phosphorylation would be to
selectively enhance the formation of protein dimer-DNA complexes at the
expense of protein monomer-DNA complexes. However, this effect was not,
in fact, that which was observed (Fig. 3). Alternatively,
phosphorylation could decrease the absolute affinity of receptor for
the DNA half-site, causing a parallel inhibition of binding of the DNA
probe by both receptor monomers and receptor dimers; however, again
this was not observed (Fig. 3). Instead, phosphorylation by PKA
selectively inhibited monomer complex formation without a significant
change in the amount of dimer complex (Fig. 3,
A-C; and quantified for the DR-4 element over a
range of receptor concentrations in Fig.
4). This selective inhibition of monomer binding by PKA, and its independence from observable effects on dimer
binding, was particularly striking on a DNA element containing only a
single half-site, which is bound exclusively by receptor monomers (Fig.
3D). We suggest that there are differences in the precise
mechanisms by which receptor monomers recognize DNA versus receptor dimers and that phosphorylation selectively inhibits DNA
recognition by receptor monomers (see "Discussion").

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Fig. 4.
PKA inhibition of DNA binding by receptor
monomers operates over a wide range of receptor concentrations.
T3R -1, prepared from baculovirus-infected Sf9 cells, was
either treated (hatched bars) or not (solid bars)
with PKA (1.4 units/reaction) and rATP for 20 min at 30 °C. The
receptor preparation was then employed in an electrophoretic gel shift
assay, using a DR-4 DNA element as the radiolabeled probe and employing
a range of receptor concentrations (as detailed below the
panel). The amount of radiolabeled probe migrating at the position of
receptor monomer (A) or receptor dimer (B) in the
resulting electrophoretogram was quantified for each receptor
concentration by PhosphorImager analysis (Molecular Dynamics Storm
system and ImageQuaNT software) and is presented in arbitrary
units.
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The above results were obtained using T3R
as the sole receptor
molecule. Notably, RXRs can dramatically enhance the ability of T3Rs to
bind to certain DNA response elements by forming receptor heterodimers;
these heterodimers display both an increased affinity for DNA, and
enhanced transcriptional regulation properties, over those observed
with the corresponding homodimers (1-8, 28). We therefore next tested
the effects of PKA on RXR/T3R
heterodimer formation. As previously
reported (29-31), addition of RXR to the T3R
preparations resulted
in the formation of a novel complex migrating at a position consistent
with that of an RXR/T3R heterodimer (Fig.
5, compare lanes 1-6 with
lanes 7-12). This RXR·T3R heterodimeric complex was
formed in preference to formation of the T3R monomer or T3R homodimer
complexes, whereas RXR itself failed to detectably bind to the DR-4 DNA
element (Fig. 5, lane 13). Prior incubation of the T3R
with PKA resulted in no detectable alteration in the binding of the DNA
by these RXR/T3R heterodimers (Fig. 5; compare PKA-treated lanes
1 and 2 to mock-treated lane 3). We conclude that PKA treatment specifically and strongly inhibits the ability of
T3R
monomers to complex with DNA, without detectably altering DNA
recognition by either T3R
homodimers or RXR/T3R
heterodimers.

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Fig. 5.
PKA treatment does not inhibit DNA binding by
RXR/T3R heterodimers. Avian T3R and RXR were tested by
electrophoretic gel shift assay for the ability to bind to a
radiolabeled DR-4 DNA probe either as separate protein preparations
(lanes 7-13) or when mixed together (lanes
1-6). The receptors were either mock-treated (lanes 3, 6, 9, 12, and 13), treated with 0.72 units of PKA
(lanes 1, 4, 7, and 10), or with 7.2 units of PKA
(lanes 2, 5, 8, and 11) in the presence (+) or
absence ( ) of rATP, as indicated below the panel. An
autoradiogram of the electrophoretogram is presented. The positions of
unbound DNA probe (Free Probe), of T3R monomers
(Mono), of T3R homodimers (Homo), and of RXR/T3R
heterodimers (Hetero) are indicated on the left
of the panel.
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Thyroid hormone destabilizes the ability of T3R homodimers,
but not monomers or RXR heterodimers, to bind to certain DNA
response elements. Notably, however, cognate T3 hormone neither
enhanced nor inhibited the effect of PKA phosphorylation when assayed
on the DR-4, TREpal, or single half-site elements employed here (data not shown).
Serine 28 and Serine 29 Mediate the Effects of PKA on Receptor DNA
Recognition--
We next altered either serine 28 or serine 29 in
T3R
individually to alanine and examined these mutant receptors for
the ability to be phosphorylated by PKA. Alanine substitution of either individual serine resulted in a partial decrease, but not a complete loss, of phosphorylation of the mutant receptor by PKA (quantified in
Fig. 6A). These results
suggested that serines 28 and 29 might both be substrates for PKA.
Supporting this hypothesis, substitution of both serine 28 and 29 to
alanines, producing a S28A/S29A double mutant, resulted in a near-total
abrogation of phosphorylation of the mutant receptor by PKA (Fig.
6A). We next examined the DNA binding properties of these
different T3R
mutants by gel mobility shift assay (Fig.
6B, and quantified in Fig. 6C). In the absence of
PKA, the S28A/S29A mutant T3R
bound to the DR-4 DNA element as a
mixed monomer/dimer population indistinguishable from that produced by
the wild-type T3R
(Fig. 6B). After treatment with PKA,
the wild-type T3R
exhibited the expected loss in monomer complex
formation, whereas the S28A/S29A double mutant fully retained the
ability to bind to the DR-4 element as both dimeric and monomeric protein complexes (Fig. 6, B and C). A similar
resistance to the effects of PKA were obtained with the S28A/S29A
double mutant when using a monomer-specific DNA probe in the binding
assay (Fig. 6D). In contrast to the double mutant, treatment
of either the S28A or the S29A single mutant with PKA produced only a
partial inhibition of monomer binding (40% and 39% inhibition
respectively, on a DR-4 element). These results suggest that the two
consecutive serine residues in avian T3R
-1 are both phosphorylated
by PKA and that both serines are likely to play a contributory role in mediating the effects of PKA on the receptor-DNA interaction.

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Fig. 6.
Mutation of both serines 28 and 29 in avian
T3R -1 abolishes phosphorylation and abolishes the PKA-mediated
phenotype. Serine 28 and 29 residues of avian T3R -1 were
mutated either individually or jointly to alanines, and the resulting
mutant proteins were tested for the ability to be phosphorylated by PKA
and to bind DNA in vitro. A N-terminal deletion mutant of
T3R was also tested in parallel. The individual S28A and S29A mutant
proteins were synthesized in a GST fusion expression system, whereas
the S28A/S29A double mutant and N-terminal deletion mutant were
synthesized in a baculovirus/Sf9 cell expression system;
comparisons were performed using wild-type T3R proteins synthesized
in the corresponding homologous expression systems. The ability of the
mutant proteins to be phosphorylated by PKA was determined by an
in vitro kinase assay and SDS-PAGE as in Fig. 2; the amount
of 32P incorporated into each mutant protein was then
quantified by PhosphorImager analysis and compared with the
32P radiolabel incorporated into the analogous wild-type
T3R under identical conditions (defined as 100%) (A).
The ability of the S28A/S29A double mutant, N-terminal deletion
mutant, or wild-type T3R protein to bind to a DR-4 DNA probe in the
presence or absence of PKA pretreatment was determined as in Fig.
3A; both an autoradiogram of the resulting electrophoretic
shift assay (B) and a quantified representation of the
amount of monomer and dimer complexes (C) are presented. A
similar assay using the single half-site 1S probe was also performed
and the results quantified (D). The amount of each complex
in the absence of PKA is defined as 100%. All treatments were
performed in the presence of rATP.
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We also tested the phosphorylation and DNA binding properties of a
T3R
mutant bearing a complete deletion of the receptor N-terminal
domain (removing amino acids 1-32). This
N-terminal deletion mutant
was not detectably phosphorylated by PKA, providing an independent
confirmation that the PKA sites lie primarily within the N-terminal
T3R
domain that encompasses serines 28 and 29 (Fig. 6A).
Intriguingly, the
N-terminal T3R
mutant bound to DNA exclusively
as a protein dimer in either the absence or the presence of PKA
treatment (Fig. 6, B and C). Our mutational
analysis therefore suggests that the N-terminal domain of avian T3R
can play an important role in DNA recognition by determining if a given
DNA sequence is bound by receptor monomers or by receptor dimers, and
that either deletion of the N-terminal domain or phosphorylation of
specific serines in this N-terminal domain by PKA can inhibit monomer
binding without precluding dimer binding. Similar results were obtained
with v-ERB A (data not shown).
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DISCUSSION |
DNA Binding by T3R
-1 Protein Monomers, but Not by Dimers, Is
Specifically Inhibited by Receptor Phosphorylation by PKA--
The
actions of a wide range of transcription factors, including the nuclear
hormone receptors, are regulated by phosphorylation (36-40, 48). We
sought to determine the effects of one particular protein kinase, PKA,
on the actions of avian T3R
-1 and its oncogenic derivative, v-ERB A. PKA phosphorylates two vicinal serines (codons 28 and 29) in the N
terminus of the T3R
receptor, and these PKA phosphorylation sites
have been previously demonstrated to be essential for the oncogenic
abilities of the v-ERB A oncoprotein and, possibly, for full
transcriptional activity by T3R
-1 (41, 42). Alterations in PKA
activity have also been reported to modify T3R
-1 activity in cells
(38, 43). We report here that PKA treatment manifests in
vitro as a selective inhibition of the ability of T3R
and v-ERB
A to bind to DNA as receptor monomers. We suggest, therefore, that
phosphorylation may also provide a means by which promoter recognition
is modulated in vivo by altering the nature of the
receptor-DNA complex. Our results are consistent with previous studies
demonstrating that the N-terminal domain, in which serines 28/29
reside, can exert profound effects on DNA recognition by the nuclear
hormone receptors and further emphasize that important elements of DNA
recognition are controlled by determinants outside the zinc finger
motif domain itself (22, 23, 25, 26).
Our basis for these conclusions is that treatment of T3R
or v-ERB A
with purified preparations of PKA resulted in a strong and selective
inhibition of receptor monomer binding to DNA, without significantly
inhibiting or enhancing the ability of the receptor to bind as a
homodimer or as a heterodimer with RXR. These effects of PKA were
observed with a variety of response elements, including a direct repeat
element, a palindromic element, and an element containing only a single
half-site. In all cases, and over a range of receptor concentrations,
only the binding of the monomer form of receptor was destabilized by
PKA treatment. No evidence was obtained that PKA phosphorylation
enhanced the intrinsic affinity of receptor for DNA; such a scenario
would predict a PKA-mediated increase in overall DNA binding by
receptor that was not observed. Nor did PKA appear to operate by
enhancing the protein-protein interactions that lead to dimer
formation; such a mechanism would require that the observed loss of
monomer binding be paralleled by an equivalent gain in dimer formation,
a phenomenon that was not observed. Instead, we consistently observed a
PKA-mediated loss of monomer binding with little or no effect on homo-
or heterodimer binding, suggesting that DNA recognition by
monomers and dimers can be independently regulated.
Our results, therefore, suggest that the mechanism of DNA recognition
by receptor dimers must be in some respects distinct from that by
receptor monomers. It is notable in this regard that codons 28 and 29 reside in a region of the receptor that participates both in the
DNA/protein contacts involved in DNA recognition and in the
protein-protein contacts involved in receptor dimerization (24).
Perhaps certain of the DNA contacts made by the T3R monomer are
pre-empted by the protein-protein contacts involved in dimerization. Phosphorylation of serine 28/29 by PKA could disrupt these
monomer-specific DNA contacts, thereby destabilizing monomer binding,
but would have little or no effect on DNA binding by the dimer. The
DNA/protein contacts made by the receptor molecules in a dimer and in a
monomer appear to be non-identical (e.g. Refs. 47 and 49),
thereby supporting our hypothesis. Our hypothesis is also consistent
with the observed lack of monomer binding by an N-terminal deletion mutant of T3R; presumably in the absence of the N terminus of the
receptor, the additional protein/DNA contacts necessary to stabilize
monomer binding cannot occur. We are currently further testing aspects
of this proposal.
Both Serine 28 and Serine 29 Play a Role in PKA Phosphorylation and
in Inhibition of Monomer Binding--
Previous studies localized the
primary site of PKA phosphorylation in avian T3R
-1 to a polypeptide
containing serines 28 and 29 (42). Intriguingly, due to the presence of
nested consensus motifs, both serine 28 and serine 29 represent
potential PKA substrates, and the precise site of modification was not
previously determined (42). Our own results strongly suggest that
serines 28 and 29 are, in fact, both targets of PKA phosphorylation
in vitro and that both sites can be phosphorylated
simultaneously. First, our measurements of stoichiometry are consistent
with a maximum of two phosphorylation sites per receptor molecule.
Furthermore, single substitutions of either serine 28 or serine 29 with
alanine reduced, but did not eliminate, T3R
phosphorylation. In
contrast, simultaneous substitution of both serines with alanines
abrogated phosphorylation. Paralleling the phosphorylation results,
single substitution of either serine reduced, but did not eliminate, the inhibitory effects of PKA on receptor monomer binding to DNA, whereas a double substitution mutant virtually completely eliminated the inhibitory effects of PKA. We conclude that either serine can be
phosphorylated by PKA and that both serines are involved in the
inhibition of monomer binding to DNA observed in the wild-type receptor.
Phosphorylation by PKA May Operate to Alter Promoter Recognition or
Utilization by Avian T3R
-1 and by v-ERB A--
Phosphorylation by
PKA (or a PKA-like kinase) appears to be crucial for the ability of
v-ERB A to participate in erythroleukemogenesis (41). The wild-type
v-ERB A protein blocks erythroid differentiation, blocks expression of
erythroid-specific genes, and allows proliferation of erythroid cells
in simple media (50, 51). In contrast, conversion of both serines 16 and 17 in v-ERB A to alanines (equivalent to serines 28 and 29 in
T3R
-1) abolished all of these v-ERB A activities, as did treatment
of wild-type v-ERB A-infected cells with a kinase inhibitor, H7.
Similarly, inducers of PKA activity such as forskolin enhance
phosphorylation of avian T3R
-1 in cells and enhance T3R
transcriptional activity, whereas inhibitors of PKA activity can
attenuate both T3R
phosphorylation and T3R
transcriptional
activity (38, 42, 52). Although some of these effects of kinase
activators or inhibitors may be indirect, the preponderance of the
evidence suggests that the phosphorylation state of v-ERB A, and likely
of T3R
, is important for function.
Despite this evidence supporting a critical role for PKA-mediated
phosphorylation in T3R and v-ERB A function, the mechanism by which the
PKA effects are mediated has remained unclear. The double alanine
mutant of v-ERB A exhibits the same apparent stability, subcellular
localization, and DNA binding (as a dimer) as does the wild-type
protein (41). Furthermore, although the double alanine substitution
fails to repress differentiation-specific genes in erythroid cells, it
nonetheless efficiently represses reporter gene transcription from a
prototypic two half-site response element in transient transfections
(41). The results we present in this article suggest a possible
explanation for these apparently contradictory results. As observed
here, PKA phosphorylation selectively inhibits DNA binding by receptor
monomers; this modification therefore has the potential to alter the
pattern of promoter recognition by receptor. This may be manifested as
preventing recognition of promoters composed of single half-sites
(single half-sites can, in fact, mediate T3R transcriptional
regulation; e.g. Refs. 32 and 53) or perhaps by altering
recognition of promotors that have odd numbers (three or more) of
half-sites, such as the malic enzyme promoter or the rat growth hormone
promoter (54, 55). Ultimately, however, more work will be required to
determine if PKA has effects on receptor function in addition to the
DNA recognition properties examined here.
Previous studies have shown that phosphorylation of T3Rs can be
mediated by a variety of different kinases and that these different
kinases can have distinct effects on T3R function (38, 41, 42, 56-58).
Both positive and negative effects on DNA recognition have been
reported. For example, a HeLa cell kinase has been identified that
enhances DNA binding by T3R dimers (47, 58), whereas the purified PKA
activity characterized here operates to destabilize DNA binding by
receptor monomers. The synergistic effects of these two kinase
activities may play a complementary role in modulating the response to
thyroid hormone under physiological conditions. Phosphorylation
inhibits DNA binding by T3R
-2, an "orphan" receptor with strong
repression properties, although the kinase involved, and the precise
effects on monomers versus dimers, have not been fully
determined (39). Furthermore, many other nuclear hormone receptors,
such as the retinoid and steroid receptors, are also targets of protein
kinase modifications and are likely to be subject to similar, multiple
levels of regulation.