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J. Biol. Chem., Vol. 276, Issue 37, 35042-35048, September 14, 2001
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From the Department of Pharmacology and Cancer Biology, Duke
University Medical Center, Durham, North Carolina 27710
Received for publication, June 4, 2001, and in revised form, July 18, 2001
In C2C12 myoblasts, endogenous histone
deacetylase HDAC4 shuttles between cytoplasmic and nuclear
compartments, supporting the hypothesis that its subcellular
localization is dynamically regulated. However, upon differentiation,
this dynamic equilibrium is disturbed and we find that HDAC4
accumulates in the nuclei of myotubes, suggesting a positive role of
nuclear HDAC4 in muscle differentiation. Consistent with the notion of
regulation of HDAC4 intracellular trafficking, we reveal that HDAC4
contains a modular structure consisting of a C-terminal autonomous
nuclear export domain, which, in conjunction with an internal
regulatory domain responsive to
calcium/calmodulin-dependent protein kinase IV (CaMKIV), determines its subcellular localization. CaMKIV phosphorylates HDAC4
in vitro and promotes its nuclear-cytoplasmic shuttling in vivo. However, although 14-3-3 binding of HDAC4 has been
proposed to be important for its cytoplasmic retention, we find this
interaction to be independent of CaMKIV. Rather, the HDAC4·14-3-3
complex exists in the nucleus and is required to confer CaMKIV
responsiveness. Our results suggest that the subcellular localization
of HDAC4 is regulated by sequential phosphorylation events. The first
event is catalyzed by a yet to be identified protein kinase that
promotes 14-3-3 binding, and the second event, involving protein
kinases such as CaMKIV, leads to efficient nuclear export of the
HDAC4·14-3-3 complex.
Accumulating evidence indicates that active transcriptional
repression is an important component of many physiological events regulated at the level of gene expression, including muscle
differentiation (1). The repression of transcription is manifest at the
level of chromatin structure where histone deacetylases
(HDACs)1 are recruited to
deacetylate histones and create a repressive chromatin structure
(reviewed in Ref. 2). Of the ten human HDACs identified so far
(3),2 HDAC4 and its closely
related family member HDAC5 have been specifically implicated in
regulating muscle differentiation ((1) and see below).
The functional link between HDAC4/5 and muscle differentiation was
first uncovered by the cloning of MITR, a transcriptional repressor
identified as an interactive partner for myocyte enhancer factor 2 (MEF-2) transcription factor family members, which are important for
muscle differentiation (4). MITR shows extensive homology to the
non-catalytic N terminus of HDAC4 and -5 (4). Indeed both HDAC4 and
HDAC5 interact with MEF-2. It was reported that overexpression of HDAC4
or HDAC5 represses MEF-2 transcriptional activity (5) and suppresses
C2C12 myoblast differentiation (1). It was also found that the
HDAC4/5·MEF-2 interaction and the effect of this complex on
muscle differentiation could be reversed by a constitutively active
form of a calcium/calmodulin-dependent protein kinase
(CaMK) (6). However, the mechanism by which CaMK regulates HDAC4 and
HDAC5 is not entirely clear.
When ectopically expressed, HDAC4 can be found in either the nucleus or
cytoplasm whereas the closely related HDAC5 is predominantly a nuclear
protein ((7) and see Fig. 1). The observed localization patterns of
different HDACs support the idea that the deacetylase activity of HDAC4
and HDAC5 might be controlled by their differential distribution in
subcellular compartments and suggest that subcellular trafficking may
be dynamically regulated. Indeed, inhibition of the nuclear export
machinery leads to nuclear accumulation of HDAC4, indicating that its
subcellular localization is controlled by active nuclear export (7).
However, nuclear export activity alone is not likely sufficient to
explain the intracellular trafficking of HDAC4, because not all cells
expressing HDAC4 show identical distribution. This suggests the
presence of other regulatory mechanisms required for the control of
HDAC4 subcellular localization. One such mechanism may involve members
of the 14-3-3 protein family, which bind a consensus motif that
contains specific phosphorylated serine residues (8). HDAC4 binds
14-3-3 family members, and the mutation of three serine residues
that abolishes 14-3-3 binding leads to nuclear accumulation of HDAC4.
Based on such studies, it was proposed that 14-3-3 traps HDAC4 in the
cytoplasm (9, 10). More importantly, the association with 14-3-3 is
sensitive to phosphatase inhibitors, suggesting that this interaction,
and therefore the subcellular distribution of HDAC4, is regulated by
reversible phosphorylation (9). This idea is supported by the
observation that the ability of HDAC4 to repress transcription can be inhibited by active forms of CaMK (6). Together, these observations suggest the possibility that subcellular localization of
HDAC4 might be regulated by CaMK-dependent phosphorylation.
Although the analysis of ectopically expressed HDAC4 suggests that its
subcellular localization is regulated by nuclear export, this has not
been shown for the endogenous protein. In this report, we present
evidence that endogenous HDAC4 shuttles between nuclear and cytoplasmic
compartments during muscle differentiation, suggesting a dynamic
regulation of HDAC4 by differential subcellular localization. Furthermore, we show that subcellular localization of HDAC4 is controlled by both a C-terminal autonomous export domain and a central
regulatory domain. We further show that the expression of an active
form of CaMKIV mobilizes HDAC4 from nucleus to cytoplasm and that HDAC4
can be phosphorylated by CaMKIV in vitro, suggesting that
CaMKIV-dependent phosphorylation can control HDAC4
subcellular localization. We present evidence that HDAC4 constitutively
binds 14-3-3 and this interaction is necessary for HDAC4 to respond to
CaMKIV. However, CaMKIV promotes HDAC4 nuclear export without stimulating HDAC4·14-3-3 interaction. These results suggest the existence of two separate phosphorylation events in which HDAC4 is
first phosphorylated by a yet to be identified kinase that creates an
HDAC4·14-3-3 complex, followed by secondary phosphorylation events,
catalyzed by kinases such as CaMKIV that activate HDAC4 nuclear export.
DNA Constructs and Antibodies--
FLAG-tagged PBJ5-HDAC4 and
HDAC5 expression plasmids were kindly provided by Dr. S. Schreiber
(Harvard University). pBJ5-HDAC4-S467A/S632A (2S/A) mutant was
generated using a QuikChange site-directed mutagenesis kit
(Stratagene). Constitutively active and inactive CaMKIV constructs were
previously described (11). 3X MEF-2 luciferase reporter construct was
kindly provided by Dr. Eric Olson (University of Texas Southwestern
Medical Center). Gal4 DNA binding domain (Gal4DBD)-HDAC4 fusion
constructs were generated in pCMX-Gal4 vector (12). Retrovirus HDAC4
and HDAC5 plasmids were made in CR-Neo retroviral vector. Anti-HDAC4
antibodies were made by injecting rabbits with recombinant HDAC4 (amino
acids 453-654) followed by affinity purification. Anti-MHC (MF20) was
used as described previously (13). Anti-FLAG (M2) antibody was
purchased from Sigma Chemical Co. Anti-Gal4DBD and anti-14-3-3
antibodies were purchased from Santa Cruz Biotechnology. Immunostaining
was performed as described previously (13).
Cell Cultures--
All cells were maintained in Dulbecco's
modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) at
37 °C in a humidified atmosphere with 10% CO2 except
for C2C12, which is maintained in DMEM with 20% FBS in 5%
CO2. To establish C2C12 stable cell lines, retroviruses
expressing HDAC4, HDAC5, or the neomycin-resistance gene were packaged
in phoenix cells. Viral supernatants were used to infect C2C12 cells in
the presence of 8 µg/ml Polybrene followed by G418 (600 µg/ml)
selection for 5 days in DMEM-20% FBS to obtain pools of stable cell
lines. Differentiation was induced when cells became 80% confluent by
replacing medium with DMEM containing 2% horse serum for 72 h.
Transfections, Co-immunoprecipitation, and Western Blot--
All
transfections were performed by the calcium phosphate method as
described previously (14). 36 h after transfection, cells were
harvested and lysed with buffer containing 0.5% Nonidet P-40, 20 mM Tris, 170 mM NaCl, 1 mM EDTA,
supplemented with 1 mM dithiothreitol and protease
inhibitors. A 200-µg aliquot of protein was immunoprecipitated by
anti-FLAG (M2) antibody-conjugated beads for 4 h followed by
immunoblotting using anti-14-3-3 antibody.
Luciferase Reporter Assay--
U2OS cells were transfected in
24-well plates with plasmids encoding 3XMEF-2 Luc reporter (0.25 µg),
internal control pCMX- In Vitro Phosphorylation Assay on Recombinant HDAC4
Fragments--
HDAC4 fragment (453) and its S/A
(S467A/S632A) mutant were cloned into pGEX-4T1 vector.
Recombinant proteins were purified by the manufacturer's recommended
protocol (Amersham Pharmacia Biotech). Glutathione
S-transferase-CaMKIV (200 ng) and maltose-binding protein-calcium/calmodulin-dependent protein kinase kinase Changes in Subcellular Localization of HDAC4 during Muscle
Differentiation--
Previous studies of ectopically expressed HDAC4
indicate that the subcellular localization of HDAC4 is regulated (7).
To begin to address the physiological importance of this regulation, we
first examined the localization of endogenous HDAC4 in C2C12 myoblasts,
because the HDAC4·MEF-2 complex has been suggested to play a role in
muscle differentiation (1). Using a highly specific antibody raised
against HDAC4 (Fig. 1N),
endogenous HDAC4 in cycling C2C12 cells is found mainly in the
cytoplasm, although a portion of the cells demonstrate nuclear staining
(Fig. 1A). Treatment of cycling C2C12 myoblasts with a
nuclear export inhibitor, leptomycin B (LMB), results in an exclusively
nuclear staining, demonstrating that endogenous HDAC4 also shuttles
between cytoplasm and nucleus via a manner that is regulated by nuclear
export (Fig. 1B). We next examined the localization of HDAC4
in terminally differentiated myotubes. As shown in Fig. 1C
(arrowheads), many differentiated C2C12 myotubes stained
with HDAC4 antibody show strong nuclear signals. These data indicate
that at least a portion of HDAC4 is transported into the nuclei of the
differentiated myotubes, which also stain positively for the muscle
differentiation marker myosin heavy chain (MHC) (Fig. 1D).
Our results suggest that the subcellular localization of HDAC4 is
dynamic and that a population of HDAC4 molecules resides in the nucleus
upon terminal differentiation.
To further evaluate the subcellular localization of HDAC4 and HDAC5
during muscle differentiation, we established C2C12 cell lines that
stably express FLAG-tagged HDAC4 or HDAC5. We stably introduced HDAC4
or HDAC5 by retrovirus-mediated gene transfer and analyzed pools of
infected C2C12 cells to avoid potential artifacts that might be
associated with a small number of selected clones. We then followed the
subcellular distribution of HDAC4 and HDAC5 in infected C2C12 myoblasts
by immunohistochemistry using antibodies against HDAC4, HDAC5, or the
FLAG epitope. C2C12 cells that stably express ectopic HDAC4 or HDAC5
display no gross abnormalities. As shown in Fig. 1E, HDAC4
is found to be more concentrated in the cytoplasm of the dividing
undifferentiated myoblasts. This cytoplasmic population of HDAC4
responds to LMB treatment and is retained in the nucleus (data not
shown). In contrast, a majority of HDAC5 is already localized in the
nucleus of the cycling C2C12 cells (Fig. 1H). This result
suggests that the subcellular localization of HDAC4 and HDAC5 may be
regulated differently. To determine if HDAC4 translocates into nuclei
upon terminal differentiation, we induced differentiation of myoblasts stably expressing HDAC4 or HDAC5. As shown in Fig. 1 (F and
G), strong nuclear staining of HDAC4 is observed in the
differentiated myotubes (arrowheads). Similar results were
obtained using anti-FLAG antibody or an HDAC4-GFP fusion protein stably
expressed in C2C12 cells (data not shown). These observations support
the idea that a portion of HDAC4 accumulates in the nucleus upon
differentiation. In fact, in some C2C12 cells, nuclear accumulation of
HDAC4 can be observed 24 h after the differentiation stimulus
(data not shown). On the other hand, HDAC5 remains concentrated in the
nuclear compartment in the differentiated myotubes (Fig. 1,
I and J). Together, our results demonstrate a
relocation of HDAC4 from cytoplasm to nucleus during muscle cell
differentiation, whereas the majority of HDAC5 appears to reside in the
nucleus in both cycling and differentiated myotubes.
To our surprise, ectopic expression of HDAC4 or -5 does not block C2C12
differentiation. The C2C12 cells containing elevated concentrations of
HDAC4 or -5 form multinucleated myotubes (Fig. 1, G,
J, L, and M) that express the
appropriate terminal differentiation marker MHC (Fig. 1, G
and J). In fact, C2C12 cells that stably express HDAC4 or -5 differentiate into large myotubes that contract vigorously. This
contraction often leads to their dislodgment from the culture dishes,
and the partially detached myotubes become ball-like structures (Fig.
1M, arrowheads). Such structures were never
observed in control C2C12 myotubes (data not shown). These observations
not only suggest that a portion of HDAC4 translocates to the nucleus in
response to differentiation but also raise the possibility that nuclear
HDAC4 and/or HDAC5 may play roles in facilitating certain aspects of
muscle differentiation.
HDAC4 Contains an Autonomous Nuclear Export Domain--
The
analysis of HDAC4 subcellular localization in C2C12 cells demonstrates
that the movement of HDAC4 occurs during muscle differentiation. To
examine the structural basis and potential factors that might regulate
this movement, we turned to U2OS cells, which are more amenable to
structure-function analysis of HDAC4 expressed by transfection. When
U2OS cells are cultured in 10% CO2, transfected HDAC4 is
localized in nuclear or cytoplasmic compartments while transfected
HDAC5 is exclusively localized in the nucleus (Fig.
2A). Similar to endogenous
HDAC4 in myoblasts, treatment with the nuclear export inhibitor LMB
leads to nuclear accumulation of the transfected HDAC4 (Fig.
2A, right panel). Thus, this assay system allows
us to study the regulation of intracellular HDAC4 shuttling.
We first investigated which region of HDAC4 is required for nuclear
export. We fused a series of HDAC4 truncation mutants to the DNA
binding domain of the yeast Gal4 transcription factor (Gal4DBD, Fig. 2B), which contains an intrinsic
nuclear localization signal and itself localizes to the nucleus (data
not shown). Analysis of the subcellular localization of these
Gal4DBD-HDAC4 fusion proteins shows that amino acids 859-1084 at the C
terminus of HDAC4 are sufficient to confer nuclear export activity and
redistribute a significant amount of nuclear Gal4DBD to the cytoplasm
(Figs. 2C, middle left panel and 2D,
upper panel). Upon LMB treatment, this fusion protein again
accumulates in the nucleus demonstrating that the intrinsic export
activity resides in fragment 859-1084 (Figs. 2C,
middle right panel and 2D, lower
panel). Supporting this conclusion, deletion of the C terminus,
which encompasses this domain, induces the nuclear localization of
HDAC4 and abrogates the LMB response (data not shown). In addition,
Gal4DBD fused with the N-terminal portion of HDAC4 (amino acids 1-223
or 222-630) is also not LMB-responsive (data not shown and Fig.
2, C and D, lower panel). Thus, the C
terminus of HDAC4 contains a domain that mediates nuclear export.
A Central Regulatory Domain Is Required for Stimulation of Nuclear
Export by CaMKIV--
The observation that phosphatase inhibitors can
affect the subcellular distribution of HDAC4 suggests that the
localization of HDAC4 should be regulated by protein phosphorylation
(9). An active form of calcium/calmodulin-dependent protein
kinase IV (CaMKIV) has been shown to be capable of regulating the
dissociation of MEF-2 from HDAC4 in vitro (6). Because
CaMKIV is present in the U2OS
cells,3 we asked whether
ectopic CaMKIV could regulate HDAC4 subcellular localization. To test
this hypothesis, we co-transfected HDAC4 with either a constitutively
active form of CaMKIV or its corresponding kinase-inactive version into
U2OS cells (11). As shown in Fig. 3A (b and
c), co-expression of active CaMKIV, but not the
kinase-inactive mutant, mobilizes nuclear HDAC4 and leads to its
accumulation in the cytoplasm. Importantly, the ability of CaMKIV to
mobilize HDAC4 is completely abrogated by LMB treatment demonstrating
that CaMKIV functions by influencing export rather than inhibiting nuclear import of HDAC4 (Fig. 3A (d)).
To map the regulatory domain in HDAC4 that is responsive to CaMK, we
determined the ability of active CaMKIV to mobilize a series of HDAC4
deletion mutants fused to Gal4DBD. As shown in Fig. 3B,
deletion of amino acids 1-221 (fragment Gal4-(222-1084)) does
not affect CaMKIV responsiveness. A further deletion to amino acid 629 (fragment Gal4-(629-1084)) eliminates the CaMKIV response, although
this fragment contains the nuclear export domain (Fig. 2). This result
indicates that amino acids in the 222-630 fragment of HDAC4 might be
regulated by CaMKIV. Consistent with the idea that CaMKIV mobilizes
HDAC4 by activating its nuclear export (Fig. 3A), this
fragment (222) alone without the C-terminal export domain (629-1084) is not responsive to CaMKIV. Together, our results suggest
that the subcellular localization of HDAC4 is regulated by two separate
domains, one of which confers autonomous export activity while the
other confers CaMKIV responsiveness.
CaMKIV-dependent Phosphorylation Induces HDAC4
Cytoplasmic Accumulation--
To determine if CaMKIV might modulate
HDAC4 subcellular localization by direct phosphorylation, we tested
whether HDAC4 is a substrate of CaMKIV. As shown in Fig.
4A, the incubation of purified
CaMKIV with a recombinant HDAC4 fragment (453) that contains part
of the putative CaMK-responsive domain results in HDAC4
phosphorylation. A previous study identified three phosphoserine
residues mediating HDAC4 and 14-3-3 interaction (9). Two of these
serine residues (Ser-467, Ser-632) are located in this domain,
and they fit the consensus phosphorylation sequence for CaMKIV
(RXX(S/T) (15)). Indeed, mutation of these two serine
residues to alanine (S/A mutant) reduces but does not abolish the
phosphorylation of HDAC4-(453-654) fragment by CaMKIV in
vitro (Fig. 4A). Thus, CaMKIV can phosphorylate HDAC4
at Ser-467 and/or Ser-632 in vitro.
To assess whether phosphorylation of Ser-467 and Ser-632 is
important for the CaMKIV responsiveness of HDAC4 in cells, we co-transfected an expression plasmid encoding the HDAC4 2S/A
(S467A/S632A) mutant with active CaMKIV and determined its subcellular
localization. As shown in Fig. 4, B and C, the
response of the 2S/A mutant to CaMKIV is markedly reduced when compared
with wild type or mutant HDAC4 with a single CaMKIV consensus site
(serine 467) mutated to alanine (data not shown).
To further assess the functional relevance of phosphorylation by CaMKIV
on the subcellular distribution of HDAC4, we examined the
transcriptional repression activity of wild type HDAC4 and its 2S/A
mutant in response to the constitutively active CaMKIV. Overexpression
of wild type HDAC4 suppresses MEF-2-dependent transcription (Fig. 4D), and this repression is reversed by co-expression
of CaMKIV. In contrast to wild type HDAC4, the 2S/A mutant effectively represses MEF-2 activity but it is more resistant to CaMKIV (Fig. 4D). Collectively, our results suggest that CaMKIV reverses
the transcriptional repression activity of HDAC4 by stimulating the mobilization of HDAC4 out of the nucleus.
HDAC4·14-3-3 Interaction Is Not Stimulated by CaMKIV But Is
Necessary for the CaMKIV Responsiveness--
Serine 467 and serine 632 residues have been shown to be involved in 14-3-3 binding (9) and
appear to be phosphorylated by CaMKIV in vitro (Fig.
4A). We reasoned that CaMKIV might phosphorylate HDAC4 and
promote its binding to 14-3-3, resulting in HDAC4 cytoplasmic accumulation. To test this, we determined the interaction between HDAC4
and 14-3-3 in the presence or absence of active CaMKIV. As shown
previously, HDAC4 binds 14-3-3 ((9) and Fig. 4E, lane 1). However, co-expression of CaMKIV does not increase HDAC4
binding to 14-3-3 (lane 2). This result suggests that CaMKIV
promotes HDAC4 nuclear export by a mechanism other than direct
modulation of HDAC4·14-3-3 interaction. Consistent with this
conclusion, both nuclear and cytoplasmic localized HDAC4 is bound to
14-3-3 (data not shown).
To further examine the role of 14-3-3 binding in CaMKIV responsiveness,
we determined the binding of the 2S/A mutant to 14-3-3. As shown in
Fig. 4E, the 2S/A mutant, which has a much reduced response
to CaMKIV (Fig. 4, B and C), also shows reduced
binding to 14-3-3 (lanes 3 and 4). Consistent
with a critical role of 14-3-3 binding in conferring CaMKIV
responsiveness, HDAC4 3S/A mutant (S246A/S467A/S632A), which does not
bind 14-3-3 (binding-deficient HDAC4), is completely resistant to
CaMKIV in both transcriptional repression and cytoplasmic translocation
assays (Fig. 4, B-D). These results suggest that the
interaction with 14-3-3 is necessary for HDAC4 to be exported in
response to active CaMKIV.
In this study, we provide evidence that two functional domains
work in concert to determine the subcellular localization of HDAC4. One
domain, which is present in the C terminus of HDAC4, provides a
constitutive nuclear export signal. This domain is presumed to tether
HDAC4 to the nuclear export receptor, Crm-1. The central regulatory
region, which confers responsiveness to CaMKIV, constitutes the second
domain critical to HDAC4 subcellular localization. Importantly, both
the central regulatory domain and the C-terminal export domain are
required for CaMKIV responsiveness. Thus, the cooperation of these two
modular domains determines the specific subcellular localization of HDAC4.
We have identified CaMKIV as one protein kinase that can regulate the
subcellular localization of HDAC4, at least in U2OS cells that contain
this enzyme. We considered two potential mechanisms that could underlie
the effects of CaMKIV on the intracellular trafficking of HDAC4. First,
as 14-3-3 binding is critical for HDAC4 subcellular localization and
two of the residues that can be phosphorylated by CaMKIV
(Ser-467 and Ser-632) also mediate 14-3-3 binding, CaMKIV
phosphorylation may result in the interaction of the exported HDAC4 to
cytoplasmic 14-3-3, which in turn, retains HDAC4 in the cytoplasm.
However, we found no evidence that CaMKIV promotes HDAC4 and 14-3-3 binding despite the fact that the interaction with 14-3-3 appears to be
critical for HDAC4 to respond to CaMKIV. Second, CaMKIV-mediated
phosphorylation of HDAC4 or its associated proteins may promote
active nuclear export of HDAC4. Consistent with this idea, the effect
of CaMKIV on HDAC4 subcellular localization can be completely abrogated
by the LMB-induced inhibition of nuclear export.
The observation that CaMKIV promotes HDAC4 nuclear export without
affecting the interaction with 14-3-3 has several implications. First,
it argues that, although CaMKIV can phosphorylate HDAC4 on serine
residues (467 and 632) that are important for 14-3-3 binding, these are
apparently not the major sites phosphorylated by CaMKIV that results in
the nuclear export of HDAC4 in cells. Rather, the CaMKIV-independent
interaction between HDAC4 and 14-3-3 strongly suggests that HDAC4 is
constitutively phosphorylated by one or more yet to be identified
kinases. The nuclear HDAC4·14-3-3 complex, which may be the "HDAC4
form" competent to respond to the active CaMKIV, can then be
efficiently exported from the nucleus in response to active
CaMKIV. Consistent with this idea, both CaMKIV and 14-3-3 can be found
in the nucleus (16, 17). Importantly, although 14-3-3 binding appears
to be essential for HDAC4 to undergo nuclear-cytoplasmic trafficking in
response to CaMKIV, this binding is clearly not required for the
nuclear import of HDAC4 and not essential for HDAC4 to function as a
transcriptional repressor (Fig. 4D).
It is possible that a CaMKIV-mediated phosphorylation event may promote
the interaction of the HDAC4·14-3-3 complex with the Crm-1 export
machinery and lead to efficient nuclear export. If HDAC4 is a direct
target of CaMKIV, 14-3-3 binding might play a permissive role to render
HDAC4 competent to respond to the nuclear export machinery upon HDAC4
phosphorylation, possibly by functioning as a scaffold protein
(reviewed in Ref. 18). Consistent with this idea, we note that in
vitro, CaMKIV can phosphorylate HDAC4 on the residues other than
the two involved in 14-3-3 binding (data not shown). Alternatively,
CaMKIV may phosphorylate other proteins important for HDAC4 subcellular
localization. For example, it has been reported that CaMKIV can
directly phosphorylate MEF-2 (19). It is possible that this
phosphorylation event might be sufficient to dissociate the
MEF-2·HDAC4 complex and mobilize HDAC4 out of the nucleus. The
identification of the "second" phosphorylation event by CaMKIV will
be key to substantiate a two-step phosphorylation model and fully
understand how HDAC4 subcellular localization is regulated.
Surprisingly, our results reveal a dramatic difference between the
subcellular localization of HDAC4 and HDAC5 despite their extensive
sequence homology. Even though both HDAC4 and HDAC5 can be regulated by
nuclear export, the subcellular localization of HDAC4 and HDAC5 is
clearly distinguishable (Fig. 1). Under the identical experimental
conditions, the majority of HDAC5 is localized in the nucleus while a
significant portion of HDAC4 is localized in the cytoplasm. Although
the exact molecular basis underlying this difference is not clear, we
have found that, similar to HDAC4, HDAC5 also contains an autonomous
export domain.4 The
regulatory domains of HDAC4 and HDAC5, however, could be differentially
phosphorylated, which then determine their ability to associate with
14-3-3 and ultimately their respective subcellular localization.
Consistent with this hypothesis, although HDAC4 binds 14-3-3 constitutively, it has been reported that ectopically expressed HDAC5
does not ((17), Fig. 4E and data not shown). Recently, it
was reported that an active form of
calcium/calmodulin-dependent protein kinase I (CaMKI) is
capable of promoting HDAC5·14-3-3 binding and leads to HDAC5 export
in cultured cells (17). Based on the two-step phosphorylation model
that we proposed for HDAC4, it is possible that active CaMKI can play
both roles for HDAC5 and induce both 14-3-3 binding as well as nuclear
export. Regardless of the details, the different subcellular
localization of HDAC4 and -5 suggests that these two closely related
proteins may be regulated by different mechanisms and consequently may
be involved in different biological functions.
By studying both endogenous and stably, ectopically expressed HDAC4 in
C2C12 cells, we have obtained evidence that subcellular localization of
HDAC4 is uniquely and dynamically regulated during muscle
differentiation. The analysis of HDAC4 and HDAC5 subcellular localization during C2C12 myoblast differentiation yielded another surprise in which we observed HDAC4 and HDAC5 to be present in nuclei
of the terminally differentiated myotubes. We have found that C2C12
cells stably producing a high level of HDAC4 or HDAC5 can
differentiate, express differentiation markers such as myogenin (data
not shown) and MHC, and fuse to form multinucleated myotubes (Fig. 1,
F-G, I-J, and L-M). In fact, we
routinely see high levels of HDAC5 and myogenin co-expressed in the
same nuclei as early as 24 h after the differentiation process is
initiated (data not shown). Importantly, the nuclear translocation (or
retention) of HDAC4 seems unlikely to be an event secondary to cell
cycle arrest, as HDAC4 remains predominantly in the cytoplasm of
serum-starved, quiescent NIH3T3
cells.5 Rather, we speculate
that HDAC4 and HDAC5 must reside in the nucleus during certain stages
of myogenesis to facilitate terminal differentiation. This hypothesis
is consistent with the observation that C2C12 cells overexpressing
HDAC4 tend to form large myotubes, which often contract vigorously in
contrast to control C2C12 cells (Fig. 1, K-M, and data not
shown). We note that our result is not in agreement with that reported
by Lu et al. (1) in which nuclear HDAC4 and HDAC5 were shown
to suppress C2C12 differentiation. This discrepancy could be due to the
levels of ectopically expressed HDAC4 and HDAC5. Because only a single
copy of infected gene is stably integrated per cell by
retrovirus-mediated gene transfer while typically multiple integration
events are achieved by transfection, it is possible that HDAC4 or -5 is
expressed at much higher levels in the transfection system used by Lu
et al. Extremely high levels of HDAC4 or -5 may dominantly
inhibit C2C12 differentiation. In any case, the presence of nuclear
HDAC4 and -5 in terminally differentiated muscle cells is not
consistent with the simple model that nuclear HDAC4 or -5 suppresses
myogenesis. It is possible that nuclear HDAC4 is required to suppress
the expression of certain genes for terminal muscle differentiation to
proceed normally. Alternatively, nuclear HDAC4 might catalyze the
deacetylation of non-histone proteins important in regulating C2C12
differentiation (20). Although further studies will be needed to
address these intriguing possibilities, our observations suggest a
complex mode of regulation of the subcellular localization of HDAC4,
which might play an important role in muscle differentiation.
In summary, we have shown that differential subcellular localization of
HDAC4 can be controlled by a series of specific phosphorylation events.
Based on our results, we propose a model (Fig. 4F) in which
HDAC4 is constitutively phosphorylated by an unknown kinase. This
phosphorylation leads to 14-3-3 binding. The nuclear HDAC4·14-3-3 complex but not HDAC4 alone can then respond to additional
phosphorylation events mediated by other kinases, such as CaMKIV, in
response to the initiation of specific signaling cascades. The second
phosphorylation event may allow the interaction of HDAC4·14-3-3 with
the Crm-1 export machinery, resulting in its efficient export from the nucleus.
We thank Drs. C. Gronzinger and S. Schreiber
for HDAC5, HDAC4, and 3S/A mutant plasmids and Dr. E. Olson for the
MEF-2 reporter plasmid. We are grateful to C. Hubbert and A. Guardiola
for critically reading the manuscript. We thank Drs. M. Yoshida and S. Kornbluth for kindly providing leptomycin B and fluorescence microscopes.
During the preparation of this manuscript, McKinsey
et al. (21) published a study describing that an active
CaMKI can induce HDAC5 nuclear export.
*
This work was supported by Damon Runyon-Walter Winchell
Cancer Foundation Grant DRS 20 (to T. P. Y.) and by National
Institutes of Health Grant HD07503 (to A. R. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, July 24, 2001, DOI 10.1074/jbc.M105086200
2
A. Guardiola, A. Ito, and T.-P. Yao, unpublished.
3
S. M. Lemrow and A. R. Means, data not shown.
4
T.-S. Liao and T.-P. Yao, unpublished observation.
5
T. A. Bolger and T.-P. Yao, unpublished observation.
The abbreviations used are:
HDAC, histone
deacetylase;
CaMKIV, calcium/calmodulin-dependent protein
kinase IV;
LMB, leptomycin B;
MEF-2, myocyte enhancer factor 2;
MHC, myosin heavy chain;
MITR, MEF-2-interacting transcription
repressor;
DBD, DNA binding domain;
DMEM, Dulbecco's modified
Eagle's medium;
FBS, fetal bovine serum.
The Modular Nature of Histone Deacetylase HDAC4 Confers
Phosphorylation-dependent Intracellular Trafficking*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Gal (0.25 µg), MEF-2D (0.25 µg), HDAC4 and
its S/A mutants (0.15 µg), and active or inactive CaMKIV (0.15 µg).
48 h after transfection, cells were collected and luciferase
activity was measured and normalized by -galactosidase activity as
previously described (14).
(500 ng) were mixed in "cold mix" containing 50 mM
Tris, 1 mM dithiothreitol, 0.1% Tween 20, 10 mM MgCl2, 2 mM CaCl2, 1 µM calmodulin, and 250 µM ATP and incubated
for 10 min at 30 °C. Then substrates (750 ng) and "hot mix"
(cold mix containing 2 µCi of [
-32P]ATP) were added
into reaction tubes, which were then incubated at 30 °C for another
10 min. The samples were separated by 10% SDS-polyacrylamide gel
electrophoresis, and gels were analyzed overnight in a PhosphorImager
(Molecular Dynamics).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (71K):
[in a new window]
Fig. 1.
Change in subcellular localization of HDAC4
in muscle differentiation. A-D, the
localization of endogenous HDAC4 in cycling cells
(A), cycling cells treated with leptomycin B (LMB, 10 ng/ml,
4 h) (B), or differentiated (C) C2C12 cells
assessed by an anti-HDAC4 antibody. Note that the staining of HDAC4 is
largely cytoplasmic (A, arrows) in cycling cells
but becomes nuclear in differentiated myotubes (C,
arrowheads), which also express the differentiation marker
MHC (D). E-J, the localization of ectopically
expressed HDAC4 (E and F) and HDAC5 (H
and I) in cycling C2C12 cells and in myotubes. Note that
some HDAC4 moves from cytoplasm (E) to nucleus
(F, arrowheads) upon differentiation. HDAC5 is
mainly nuclear in both cycling cells (H) and in myotubes
(I, arrowheads). Anti-MHC antibody (G
and J) was used to verify muscle differentiation.
K-M, morphological changes in C2C12 cells stably expressing
HDAC4 before (K), 48 h after (L), or 72 h after (M) differentiation was initiated. Note the presence
of multinucleated myotubes (L, arrows) and the
large number of contractile, ball-like structures (M,
arrowheads) after differentiation was initiated.
N, the specificity of anti-HDAC4 antibody. 40 µg of total
cellular protein from C2C12 cells was loaded in SDS-polyacrylamide gel
electrophoresis, and a Western blot was carried out using anti-HDAC4
antibody. A single band corresponding to endogenous HDAC4 supports the
specificity of this antibody.
View larger version (57K):
[in a new window]
Fig. 2.
Mapping the subcellular shuttling
domain on HDAC4. A, the localization of exogenous
HDAC4, HDAC4 treated with LMB (10 ng/ml) for 4 h, and HDAC5 by
immunostaining. Note the obvious difference in localization pattern
between HDAC4 and HDAC5. B, schematic diagram of various
HDAC4 fragments fused with Gal4DBD and their response to LMB.
C and D, the subcellular localization and the LMB
response of transfected Gal4-wild type (wt) HDAC4,
Gal4-(222-630), Gal4-(629-1084), and Gal4-(859-1084) as determined
by immunostaining using anti-Gal4DBD antibody. 200 cells from each
transfection were scored for the presence of the HDAC4 fragments
in cytoplasm (D, upper panel) or nucleus
(D, lower panel). The lower panel also
illustrates the effect of LMB on nuclear retention of the
fragments.
View larger version (44K):
[in a new window]
Fig. 3.
Subcellular localization of HDAC4 is
regulated by CaMKIV. In A, left panel,
FLAG-tagged HDAC4 expression plasmid was cotransfected into U2OS cells
together with an active CaMKIV (ACaMKIV) (b,
d), or inactive CaMKIV (ICaMKIV) (c)
as indicated. Cells were either untreated (a-c) or treated
(d) with LMB. The localization of HDAC4 was determined by
immunostaining as described above. Note that co-expression of CaMKIV
mobilizes HDAC4 out of the nucleus, and this effect is completely
inhibited by LMB treatment. Right panel: Quantification of
HDAC4 staining. B, mapping the region of HDAC4 required for
CaMKIV responsiveness. U2OS cells were transfected with expression
plasmids for various Gal4-HDAC4 fragments with or without the
co-expression of an activated CaMKIV as indicated. Localization of
various Gal4-HDAC4 fragments in different subcellular compartments was
determined by immunostaining using anti-Gal4DBD antibody, and the
results were quantified.
View larger version (35K):
[in a new window]
Fig. 4.
CaMKIV responsiveness and 14-3-3 binding. A, in vitro phosphorylation
of the recombinant HDAC4-(453-654) fragment and its S/A (S467A/S632A)
mutant. The degree of phosphorylation is quantified as a percentage of
relative intensities obtained by phosphorimaging and Coomassie blue
staining bands, where the wild type is set at 100%. B and
C, the subcellular localization of wild type HDAC4, 2S/A
(S467A/S632A), and 3S/A (S246/467/632A) mutants in the presence or
absence of an active CaMKIV. Note that wild type HDAC4 is exported to
cytoplasm in the presence of CaMKIV, whereas the 2S/A mutant has little
and 3S/A has no response to the active CaMKIV and remain in the
nuclei. D, transcriptional repression of a MEF-2 reporter
gene by wild type HDAC4 and S/A mutants. U2OS cells were transfected
with expression plasmids as indicated. MEF-2-dependent
luciferase activity was measured and normalized. Note that both 2S/A
and 3S/A mutants are more resistant to CaMKIV. E, binding of
14-3-3 to wild type and 2S/A HDAC4. Transfected HDAC4 and 2S/A mutant
were immunoprecipitated by anti-FLAG antibody, and the HDAC4-associated
14-3-3 was determined by Western blot with an anti-14-3-3 antibody.
F, a model for HDAC4 nuclear export. Nuclear HDAC4
(filled circles) is constitutively phosphorylated by a yet
to be identified kinase (X), which results in the formation
of an HDAC4·14-3-3 complex (filled squares).
HDAC4·14-3-3 is not efficiently exported but is competent to respond
to a subsequent phosphorylation event mediated by CaMKIV. The second
phosphorylation event triggers efficient nuclear export of the
HDAC4·14-3-3 complex by promoting its interaction with the Crm-1
export machinery via an intermediate protein (hatched
circle). P represents phosphorylation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
Addendum
FOOTNOTES
To whom correspondence should be addressed: Dept. of Pharmacology
and Cancer Biology, P. O. Box 3813, Duke University Medical Center,
Durham, NC 27710. Tel.: 919-613-8654; Fax: 919-681-8461; E-mail:
yao00001@mc.duke.edu.
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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