HDAC3 as a Molecular Chaperone for Shuttling Phosphorylated TR2 to PML: A Novel Deacetylase Activity-Independent Function of HDAC3
Wei L-N (2009) HDAC3 as a Molecular Chaperone for Shuttling Phosphorylated TR2 to PML: A Novel Deacetylase
Activity-Independent Function of HDAC3. PLoS ONE 4(2): e4363. doi:10.1371/journal.pone.0004363
HDAC3 as a Molecular Chaperone for Shuttling Phosphorylated TR2 to PML: A Novel Deacetylase Activity-Independent Function of HDAC3
Pawan Gupta 0
Ping-Chih Ho 0
Sung Gil Ha 0
Yi-Wei Lin 0
Li-Na Wei 0
Andreas Bergmann, UT MD Anderson Cancer Center, United States of America
0 1 Department of Pharmacology, University of Minnesota Medical School , Minneapolis , Minnesota, United States of America, 2 Institute of Microbial Technology , Chandigarh , India
TR2 is an orphan nuclear receptor specifically expressed in early embryos (Wei and Hsu, 1994), and a transcription factor for transcriptional regulation of important genes in stem cells including the gate keeper Oct4 (Park et al. 2007). TR2 is known to function as an activator (Wei et al. 2000), or a repressor (Chinpaisal et al., 1998, Gupta et al. 2007). Due to the lack of specific ligands, mechanisms triggering its activator or repressor function have remained puzzling for decades. Recently, we found that all-trans retinoic acid (atRA) triggers the activation of extracellular-signal-regulated kinase 2 (ERK2), which phosphorylates TR2 and stimulates its partitioning to promyelocytic leukemia (PML) nuclear bodies, thereby converting the activator function of TR2 into repression (Gupta et al. 2008; Park et al. 2007). Recruitment of TR2 to PML is a crucial step in the conversion of TR2 from an activator to a repressor. However, it is unclear how phosphorylated TR2 is recruited to PML, an essential step in converting TR2 from an activator to a repressor. In the present study, we use both in vitro and in vivo systems to address the problem of recruiting TR2 to PML nuclear bodies. First, we identify histone deacetylase 3 (HDAC3) as an effector molecule. HDAC3 is known to interact with TR2 (Franco et al. 2001) and this interaction is enhanced by the atRAstimulated phosphorylation of TR2 at Thr-210 (Gupta et al. 2008). Secondly, in this study, we also find that the carrier function of HDAC3 is independent of its deacetylase activity. Thirdly, we find another novel activity of atRA that stimulates nuclear enrichment of HDAC3 to form nuclear complex with PML, which is ERK2 independent. This is the first report identifying a deacetylase-independent function for HDAC3, which serves as a specific carrier molecule that targets a specifically phosphorylated protein to PML NBs. This is also the first study delineating how protein recruitment to PML nuclear bodies occurs, which can be stimulated by atRA in an ERK2-independent manner. These findings could provide new insights into the development of potential therapeutics and in understanding how orphan nuclear receptor activities can be regulated without ligands.
Funding: This work was supported by NIH grants DK54733, DK60521, DA11190, DA 11806 and K02-DA13926 to LNW. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Histone deacetylases (HDACs) are assigned to three distinct
classes based on their sequence similarity to the yeast RPD3 (class I),
HDA1 (class II), or Sir2 (NAD-dependent) proteins . Class I
includes HDACs 1, 2, 3, 8, and possibly 11 [1,4]. Class II includes
HDACs 4, 5, 6, 7, 9, and 10 . HDACs generally are found in
large, multiprotein corepressor complexes that are targeted to
chromatin by sequence-specific DNA-binding proteins and are
involved in repressing transcription [5,6]. These DNA-binding
proteins include nuclear receptors, the E-box binding proteins, and
the methylcytosine-binding protein MeCP2 . HDACs and
histone acetyl transferases play crucial roles in regulating histone
acetylation to regulate gene transcription .
Although the different classes of HDACs share a certain degree of
sequence homology, they exhibit different specificities in various
systems . Class I HDACs function in developing embryos
and carcinomas . HDAC3, a class I HDAC , is usually
found in corepressor complexes such as N-CoR, SMRT and RIP140
complexes . A thorough characterization of the structural
and functional properties of HDAC3 identified a nonconserved
region in its carboxy-terminal region that is required for histone
deacetylation and transcriptional repression [13,26]. It was suggested
that this carboxy-terminal domain acts in concert with the putative
catalytic domain of the protein. In addition, a nuclear export signal is
present in the central portion of the molecule (amino acids [aa] 180
330), and a nuclear localization signal is present in the
carboxyterminal region (aa 313428) .
Although HDAC3 functions primarily in histone deacetylation
, a growing list of its nonhistone substrates suggests a role for
HDAC3 in biological processes beyond transcriptional repression
[13,2729]. Studies have suggested that HDAC3 associates with
complexes that are not directly involved in regulating genome
activity , but it is not known if formation of these complexes
requires its deacetylase activity. HDAC3 has been shown to
function as a carrier/bridging molecule, targeting RbAp48 to the
retinoblastoma protein . HDAC3 also localizes to the mitotic
spindle and is required for kinetochoremicrotubule attachment
. It is presumed that most of the pathways involving HDACs
require deacetylase activity  but this has not been
demonstrated conclusively . We previously demonstrated that
HDACs 3 and 4 interact constitutively and directly with the
orphan nuclear receptor TR2 via its DNA-binding domain .
We also reported that ERK2-phosphorylated TR2 is recruited to
PML nuclear bodies (PML NBs) for its subsequent small
ubiquitinlike modification (SUMOylation) and function as a potent
transcriptional repressor [32,33]. An important question that
remains to be answered is how phosphorylated TR2 is facilitated
to the PML NBs.
TR2 is specifically expressed in early embryos , and is a
transcription factor for transcriptional regulation of important genes
in stem cells including the gate keeper Oct4 . It is known to
function as an activator , or a repressor [36,37]. Due to the lack
of specific ligands, mechanisms triggering its activator or repressor
function remains puzzling for decades. Recently, our finding of
alltrans retinoic acid (atRA) triggered phosphorylation of TR2
stimulates its partitioning to PML nuclear bodies, which converts
the activator function of TR2 into a repressor [32,33], prompted us
to examine how phosphorylated TR2 is recruited to PML, an
essential step in converting TR2 from an activator to a repressor.
In the present study we demonstrate that the interaction of
HDAC3 with TR2 can be stimulated by phosphorylation of TR2
at a specific ERK2 target. Furthermore, HDAC3 serves to target
this specifically phosphorylated TR2 to PML NBs for its
subsequent SUMOylation. Importantly, this novel function of
HDAC3 is independent of its deacetylase activity. Finally, a novel
ERK2-independent activity of atRA is identified, which stimulates
translocation and nuclear enrichment of HDAC3 to form nuclear
complex with PML.
The effect of specific TR2 phosphorylation on its
interaction with HDAC3 and PML
Our recent report  showed that site-specific phosphorylation
of TR2 at Thr-210 modulates its association with the effector
molecules PCAF and RIP140, albeit indirectly by increased
SUMOylation of TR2 at Lys-238. Because HDAC3 binds to TR2
at the hinge region that encompasses Thr-210 , we assessed its
ability to mediate recruitment of Thr-210-phosphorylated TR2 to
PML using a two-hybrid interaction test (Fig. 1A). The wild type
(WT) TR2 interacted effectively with HDAC3 . This interaction
was enhanced significantly in the phosphomimetic mutant TR2
(210CP; ThrRGlu [38,39]), but was abolished in the
phosphorylation-negative mutant TR2 (210CN; ThrRAla). Furthermore,
although the SUMOylation-defective mutant TR2 (K238R;
LysRArg) exhibited a basal level of interaction with HDAC3 similar to
that of WT TR2, a double mutant containing both the
phosphomimetic and the SUMOylation-negative mutations
(210CP+K238R) behaved like the phosphomimetic CP210 TR2 in
terms of its ability to interact with HDAC3. In contrast, a constitutive
TR2 double mutant negative for both phosphorylation and
SUMOylation (210CN+K238R) could not effectively interact with
HDACs. These data suggest that the interaction of TR2 with
HDAC3 is phosphorylation-dependent but
SUMOylation-independent. This phosphorylation-dependent interaction was verified using
pharmacological agents to activate or inactivate ERK (Fig. 1B). A
mitogen-activated protein kinase (MAPK)/ERK activator
sphingosine-1-phosphate (S-1-P) increased the association of TR2 with
HDAC3, whereas addition of an ERK inhibitor
3-(2-aminoethyl)5([4-ethoxyphenyl]methylene)-2,4-thiazolidinedione HCl (AMTZD)
completely abolished the effect.
We then investigated the relationship between Thr-210
phosphorylation or Lys-238 SUMOylation and the recruitment
of TR2 to endogenous HDAC3. Both WT FLAG-TR2 and the
phosphomimetic and deSUMOylated FLAG-210CP+K238R
TR2 double mutant associated effectively with HDAC3 (Fig. 1C).
However, the ability of TR2 to associate with HDAC3 was
abolished completely in the FLAG-210CN+K238R double
mutant defective for both phosphorylation and SUMOylation.
As predicted, the ability of TR2 to associate with PML mirrored
the pattern of TR2 association with HDAC3. Thus,
phosphorylation on Thr-210, but not deSUMOylation on Lys-238, triggers
effective association of TR2 with HDAC3 and PML.
In vitro proteinprotein interaction tests were undertaken to
determine if the association of TR2 to the effector molecule HDAC3
was direct or indirect (Fig. 1D). TR2 was expressed and purified as a
GST fusion protein. GST pull-down assays of WT and CP TR2 were
carried out using in vitro-transcribed and translated HDAC3 or PML.
WT or CP TR2 did not interact with PML (Fig. 1D, right panel), but
weak interactions between HDAC3 and WT TR2 were detected; this
effect was enhanced in the CP mutant (Fig. 1D, left panel). These data
are consistent with the results of the cell-based assay, further
supporting a phosphorylation-dependent, direct interaction between
TR2 and HDAC3. This suggests that HDACs might function as a
chaperone for the association of TR2 with PML, the target site for
TR2 SUMOylation and its conversion into a repressor.
A functional role for HDAC3 in targeting phosphorylated
TR2 to PML
Because the association of TR2 with HDAC3/PML was related
directly to phosphorylation on TR2 at Thr-210, and Thr-210
phosphorylation was a direct result of atRA stimulation, we
monitored the role of endogenous HDAC3 in mediating
atRAtriggered TR2/PML colocalization [32,33]. SiRNA knockdown of
HDAC3 (Fig. 2A, panel 4) effectively (90%) blocked the
atRAtriggered association of TR2 with PML (panel 1). This was similar
to the efficiency achieved by TR2 knockdown (92%; panel 3).
However unlike the HDAC3 knockdown, the TR2 knockdown did
not affect complex formation between HDAC3 and PML. This
suggests that TR2 does not alter the direct binding of HDAC3 to
PML, as reported previously . However, it supports the
hypothesis that HDAC3 functions as a carrier or chaperone in the
mobilization of TR2 to PML.
The role of HDAC3s deacetylase activity in facilitating TR2/
PML colocalization was examined using the deacetylase activity
inhibitor TSA (Fig. 2B, upper panel). Interestingly, blocking the
deacetylase activity of HDAC3 did not affect the atRA-triggered
TR2/PML colocalization, suggesting that the carrier role of HDAC3
was independent of its deacetylase activity. However, TSA is known
for many non-target effects , including modulation of global gene
expression. We therefore sought to validate the results of these
pharmacological studies by using a dominant negative mutant of
HDAC3 (Ser-424RAla) which is specifically defective in its
deacetylase activity . There was no apparent difference between
the WT and deacetylase-negative mutant in the ability of atRA to
stimulate the association of TR2 with PML (Fig. 2B, lower panel).
This confirms a deacetylase-independent chaperone role for HDAC3
in stimulating TR2 localization to PML.
Immunohistochemistry was also conducted to monitor the
distribution of endogenous TR2 and PML NBs in a gain- or
lossof-HDAC3-expression system (Fig. 2C). Without atRA (control
cells), TR2 was only minimally (20%) colocalized with endogenous
PML NBs. In the atRA-treated culture, 60% of the cells showed
colocalization, as reported previously . In a gain-of-function
system that acquired ectopic expression of HDAC3, TR2
Figure 2. HDAC3s role as a chaperone in TR2 partitioning to PML is independent of its deacetylase activity. (A) HDAC3 modulates TR2
partitioning to PML. HDAC3 and TR2 were silenced by RNAi in embryonic stem cells and their complex formation with PML was monitored by
coimmunoprecipitation. (B) HDAC3 chaperoning of TR2 to PML independent of deacetylase activity. TSA (a deacetylase inhibitor) was tested for its
ability to modulate atRA-triggered TR2 and PML colocalization (upper panel). Dominant negative HDAC3 was compared to WT HDAC3 for
modulation of atRA-triggered association of TR2 with PML (lower panel). (C) Immunostaining of endogenous TR2 and PML in control cells, or cells
treated with atRA (0.1 mM) for 6 h in the context of gain or loss of HDAC3 expression. Nuclei were stained with DAPI. Large colocalized TR2/PML
puncta in stimulated cells are marked with arrows. Right panel: Statistical analysis of the percentage of cells with colocalized TR2 and PML puncta
(positive) among the total cells (positive+negative).
colocalization with PML was enhanced in both the presence and
absence of atRA (60% of the cells). The enhanced,
atRAindependent, increase in TR2 recruitment to PML might have
been caused by saturation of the endogenous components. In
contrast, in a loss-of-function system where endogenous HDAC3
was knocked down, atRA-stimulated association of TR2 with
PML NBs was almost completely abolished. These results further
support a functional role for HDAC3 in atRA-stimulated
recruitment of TR2 to PML in this experimental system.
Effect of ERK and atRA on complex formation of TR2/
Because HDAC3 binds both TR2 and PML, it might act as a
chaperone for TR2 to PML. atRA activates ERK2, which
phosphorylates TR2 at Thr-210. Phosphorylated TR2 then strongly
interacts with HDAC3. To verify this model, we manipulated the
experimental system with regard to two critical elements: atRA and
ERK2 (Fig. 3). In the presence of an ERK2 inhibitor (AMTZD),
formation of the TR2HDAC3 complex was reduced (Fig. 3A, top
panel, lane c). Furthermore, because atRA phosphorylates TR2
through the ERK pathway , it was unable to rescue complex
formation (lane d). The direct interaction between HDAC3 and PML
 was increased slightly by atRA treatment (panel 2, lane b), but
ERK2 inhibition did not affect this interaction (lane d). In contrast,
the atRA-triggered association of TR2 with PML (panel 3) was
completely blocked by inhibiting ERK2 activity (lane d). Taken
together, our data suggest two independent pathways. In the first,
TR2 recruitment to HDAC3 is atRA-dependent and
ERK2sensitive, whereas the second pathway (in which HDAC3 recruitment
to PML is slightly enhanced) is responsive to atRA but is independent
Figure 3. Effect of atRA and ERK on endogenous complexes with HDAC3. (A) ERK2-dependent association of HDAC3 to TR2 but not PML.
ERK2 was inactivated pharmacologically and the atRA-triggered complex formation of HDAC3 with TR2 and PML was monitored. (B) Effects of atRA
and ERK2 on HDAC-PML complex formation and HDAC3 subcellular distribution. Nuclear (Nuc)/cytoplasmic (Cyt) distribution of HDAC3 and its
association with the import (Importin b; Karyopherin 1) or the export (exportin 1, CRM1) machinery were monitored in coimmunoprecipitation (IP)
experiments. Immuno blot (IB) panels show all input and protein controls. Numbers above the panels indicate quantified relative values. (C)
atRAtriggered nuclear enrichment of HDAC3 in P19 cells, monitored by immunocytochemistry. (D) A kinetic study of endogenous components in P19 cells
after atRA treatment at 0, 2, 8, 16, 24, and 48 h (panels 15).
of ERK2 activation (see the current working model shown in Fig. 4).
It is possible that TR2HDAC3 and HDAC3PML could exist as
two separate complexes because HDAC3PML was formed even in
the absence of TR2HDAC3 (i.e., when the culture was stimulated
with atRA but inhibited by an ERK2 inhibitor).
It is known that endogenous HDAC3 is generally distributed in
equilibrium between the cytoplasm and the nucleus [26,30].
Further, HDAC3 and PML can directly interact with each other
 and this complex formation is not dependent upon TR2
(shown above). We therefore suspected a possibility that atRA
could modulate subcellular distribution of HDAC3, thereby
enhancing its nuclear distribution. To examine this possibility
and to determine if this particular effect of atRA (stimulation of
HDAC3-PML complex formation in the nucleus) was dependent
upon the ERK2 pathway, we conducted siRNA-mediated
knockdown of ERK2 and monitored subcellular distribution of
HDAC3 and formation of HDAC-PML complex (Fig. 3B). It
appeared that HDAC3 was indeed detected in both the cytoplasm
and the nucleus in normal culture; whereas atRA treatment
stimulated significant nuclear enrichment of HDAC3 (comparing
fractions of Nuc and Cyt). Interestingly, this particular effect of
atRA on HDAC3 nuclear enrichment was not blocked by the
addition of siRNA to ERK2 (RNAi). This result suggests that the
equilibrium of HDAC3 has shifted in favor of its nuclear
localization, and that this phenomenon is ERK2 independent.
Thus, the effect of atRA on HDAC3 nuclear enrichment is clearly
different from its effect on TR2 phsophorylation that is
ERK2dependent as shown in our previous study.
To gain insights into the molecular mechanism of atRA-triggered
HDAC3 nuclear enrichment, we monitored in vivo interaction of
HDAC3 with exportin (the principal export machinery, CRM1) and
importin-b1 (karyopherin b1) (Fig. 3B). Interestingly, in atR treated
cultures, no change was detected in the interaction of HDAC3 with
importin-b1 but a clear reduction was detected in its interaction with
exportin 1. This suggests that atRA changes the equilibrium of
HDAC3, in favor of its nuclear retention, probably due to a selective
blockage of its export machinery. Other studies using
immunocytochemistry has shown HDAC3 localization mainly in many fine
punctate foci of the cytoplasm and in a diffused pattern in the
nucleus [26,30]. We also observed a similar nuclear and cytoplasmic
pattern of HDAC3 in the control cultures, but a nucleus-enriched
pattern in cultures treated with atRA (Fig. 3C). This validates the
biochemical data shown in Fig. 3B. Therefore, the result showing
atRA-stimulated nuclear enrichment of HDAC3 provides some
explanation for the increased nuclear HDAC3-PML complex
formation. However, how atRA stimulates nuclear retention, or
enrichment in the nucleus, of HDAC3 remains to be investigated.
This is indicated with a question mark in our current working model
shown in Fig. 4.
We also examined the kinetics of endogenous complex formation
at different time points following atRA treatment (Fig. 3D). The
phosphorylation of TR2 at Thr-210 was rapidly stimulated, but was
diminished by 16 h (panel 2). TR2 association with HDAC3 was
consistently apparent at 2 h and subsided after 16 h (panel 1). The
kinetics of TR2 recruitment to PML (panel 3) and its subsequent
SUMOylation (; panel 4) followed closely that of
atRAstimulated TR2 phosphorylation and HDAC3 association. Among
the HDACs examined (HDACs 16), only HDAC3 showed
detectable levels of expression in the P19 culture system (panel 5).
The results obtained with this system that contains no foreign
constructs validate our proposed model (Fig. 4): atRA stimulates
TR2 localization to PML, where SUMOylation of TR2 converts it
into an effective repressor of the Oct4 gene . The present study
has identified HDAC3 as an important carrier for the transport of
TR2 phosphorylated specifically in its DNA-binding domain to
PML NBs, a key step in the conversion of TR2s activity.
In the P19 stem cell differentiation model, atRA reduces Oct4
expression. Our previous studies reported a non-genomic
mechanism by which atRA stimulates TR2 SUMOylation, a key step in the
conversion of TR2 into a repressor of the Oct4 gene. In these earlier
studies, we confirmed that atRA rapidly activates ERK2, leading to
TR2 phosphorylation at Thr-210 and subsequent recruitment to
PML NBs for SUMOylation [32,33]. However, we failed to detect a
direct interaction of TR2 with PML (; Fig. 1D), leaving
unanswered the means by which phosphorylated TR2 was recruited
to PML. In this current study, we are able to identify this missing
link: HDAC3 interacts with TR2 in an atRA-enhanced manner.
More importantly, the carrier/chaperone function of HDAC3 in this
pathway appears to be independent of its deacetylase activity.
However, in this newly identified signaling pathway, some
outstanding questions have to be addressed as pointed out in our
current working model (Fig. 4). Apparently, questions remain that
how atRA activates ERK2, and how atRA stimulates HDAC3
shuttling to PML at the molecular level.
HDAC3 interacts directly with the DNA-binding domain of
TR2 that encompasses Thr-210 , and this interaction is
enhanced by specific phosphorylation on Thr-210 (Fig. 1D). The
exact mechanism of this enhancement remains unclear.
Presumably, phosphorylation could induce conformational changes in
TR2 such that it is more likely to interact with the binding domain
of HDAC3. The presumed direct interaction between HDAC3
and PML  is increased slightly by atRA treatment, but this is
ERK-independent. This remains an interesting point for further
investigation. Another important finding of this study is that
HDAC3 function in this pathway is independent of its deacetylase
activity. Although this observation was made using the TR2
system, HDAC3 can also interact with other proteins, including
many nuclear receptors that all harbor a very similar
DNAbinding domain. Whether HDAC3 can have a similar role in
recruiting other nuclear receptors remains to be determined.
HDAC3 associates with the nuclear dots that encompass PML
NBs [40,43,44]. It also associates with the E3 ligase PIAS in the
nuclear dots, an association that relieves the transcriptional
repression exerted by HDAC3 during TFII-I mediated gene
activation . It is unclear whether the effect of HDAC3 in this
system involves deacetylase activity . Relevant to this debate,
our current findings present evidence for a
deacetylase-independent functional role for HDAC3 in mediating molecular
interactions. The functional role of HDAC3 in leukemia has been
attributed to its association with the PMLRAR fusion protein in a
deacetylase-dependent fashion [13,41]. A variety of therapeutic
agents are being tested, targeting HDACs deacetylase activity.
The present study would suggest a new potential target that is
independent of such activity.
Materials and Methods
Plasmid Constructs and RNA interference
Mouse complementary DNAs for TR2, HDAC3, and GAL4
tkluciferase reporter were as described previously [27,33].
Constitutive negative/positive, point/sequential mutations involving
residues Thr-210, and Lys-238 in WT TR2 (CMV/FLAG/
GAL4) vector as template were made according to QuikChange
XL site-directed mutagenesis kit (Stratagene) as described
previously [PNAS]. HDAC3 enzymatically defective mutant
Ser424RAla was as described previously . Scrambled RNA and
siRNAs for Nr2c1, encoding TR2 were from Dharmacon and
siRNAs for Mapk1, encoding ERK2 were from Qiagen as
described previously . siRNA for Hdac3, encoding HDAC3
were from Qiagen 59-AGAAGAUGAUCGUCUUCAA-39 and
59-GAUGAUGAUGUGUAUAAUA-39. RNAs were introduced
using DharmaFECT1 (T-200101, Dharmacon) or HiPerfect (no.
301704, Qiagen). Silencing was assessed by Western blots at 48
72 h. Transfection and reporter assay were as described [33,42].
Assays were conducted at 1636 h.
Chemicals and Treatments
All treatments were done in Dulbeccos modified Eagles medium
containing DCC serum. atRA (0.1 mM) was added for 6 h before
harvesting, unless mentioned other wise. Activators/inhibitors
(Calbiochem) of ERK, sphingosine-1-phosphate (MAPK/ERK
activator, 1 mM),
3-(2-aminoethyl)5-([4-ethoxyphenyl]methylene)2,4-thiazolidinedione, HCl (ERK2 inhibitor, 25 mM), 5-(2
(ERK1/2 inhibitor, 1 mM) were added for 6 h. TSA (Calbiochem)
 a deacetylase activity inhibitor, was added 12 h before and
alongwith atRA treatment.
Subsequent to ectopic expression/knockdown of HDAC3
(48 h), atRA treatment was done for 6 h. Cells were fixed in 4%
formaldehyde and a permeation buffer, blocked for 30 mins, and
incubated with primary antibodies at 4uC overnight, followed by
secondary fluorescence-conjugated antibodies, at room
temperature for 3 h . Images were acquired with a fluorview confocal
system (Olympus). Quantification was conducted by scoring the
positive cells (showing 20% colocalized PML with TR2) versus
total cell numbers.
Immunoprecipitation and Western Blot Analysis
As described previously [32,33]. Antibodies were FLAG-M2
(F3165) from Sigma, phosphothreonine (ab-9337) from Abcam,
PML (05718) and HDAC3 from Upstate Biotechnology (05-813)
and Santa Cruz Biotechnology (sc11417). TR2 (sc-9087), and
PML (sc-5621) were from Santa Cruz Biotechnology. ERK2
(9108), and phosphor-p42/p44 ERK (9101) were from Cell
Signaling. Exportin 1 (CRM1) and importin-b1 (karyopherin b1)
were as described previously 
We thank the efforts of Justin Reed.
Conceived and designed the experiments: PG PCH LNW. Performed the
experiments: PG PCH SGH YWL. Analyzed the data: PG LNW.
Contributed reagents/materials/analysis tools: LNW. Wrote the paper:
PG LNW. Financial support: LNW.
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