Nucleocytoplasmic shuttling and phosphorylation of BMAL1

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Circadian
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Tamaru
Blackwell
Oxford,
Genes
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1356-9597
2003 Article
BMAL1
et al.
Blackwell
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Cells nucleocytoplasmic
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Publishing
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Ltd shuttling

are regulated by circadian clock in cultured fibroblasts

Teruya Tamaru1,*, Yasushi Isojima2, Gijsbertus T. J. van der Horst3, Kohtaro Takei4,
Katsuya Nagai2 and Ken Takamatsu1
1
Department of Physiology, Toho University School of Medicine, 5-21-16 Ohmori-nishi Ohta-ku, Tokyo 143-8540, Japan
2
Division of Protein Metabolism, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
3
Department of Cell Biology & Genetics, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, the Netherlands
4
Department of Molecular Pharmacology and Neurobiology,Yokohama City University School of Medicine, 3-9 Hukuura, Kanazawa-ku,
Yokohama 236-0004, Japan

Abstract
Background: Recent discoveries of clock proteins
have unveiled an important part of the mammalian
circadian clock mechanism. However, the molecular
clockwork that cause these fundamental feedback
loops to stably oscillate with a ∼24 h-periodicity
remain unclear.
Results: Serum-shocked fibroblasts were used as a
cellular clock model. Circadian changes in the sub-
cellular localization and phosphorylation of BMAL1
protein in these cells were assessed by immunocyto-
chemistry and immunoblotting. A significant time
lag between Bmal1 transcription and the cytoplasmic/
nuclear accumulation of BMAL1 was observed. After
its nuclear accumulation, BMAL1 accumulated in
the cytoplasm again, mainly by nucleoexport, before
the increase of Bmal1 transcripts. Nuclear accumulation
of BMAL1 matched nuclear accumulation of
CLOCK and the peak of Per1 transcription. Nuclear
BMAL1 was gradually phosphorylated and then
dephosphorylated in a temporally regulated manner,
although cytoplasmic BMAL1 was not. In serum-
shocked mCry1/mCry2 (CRY)-deficient fibroblasts,
which lack a functional clock, both the cytoplasmic
and nuclear BMAL1 were only present as hyper-
phosphorylated forms and their circadian nucleocy-
toplasmic shuttling was absent.
Conclusions: We propose that the nucleocytoplasmic
shuttling and phosphorylation states of BMAL1 are
regulated by circadian clock, and that this tempo-
rally regulated and time-delayed nuclear entry of
BMAL1 is important in the maintenance of a stably
oscillating clock.

genes in a variety of other organisms (Crosthwaite et al.

Introduction
Recent developments have unveiled an important part
of the mechanism behind circadian clocks, namely, that
a set of mammalian circadian clock genes, such as Per1,
Per2, Cry1, Cry2, Clock and Bmal1, are expressed in an
oscillatory manner with a circadian rhythm in the central
circadian pacemaker in the hypothalamic suprachiasmatic
nucleus (SCN) (Moore 1997; Morse & Sassone-Corsi
2002; Reppert & Weaver 2002). It is believed that these
mammalian clock genes may directly or indirectly regu-
late their own expression by means of feedback loops,
which is similar to what has been observed for the clock
Communicated by : Eisuke Nishida
*Correspondence: E-mail: tetamaru@med.toho-u.a.jp
DOI: 10.1046/j.1365-2443.2003.00686.x
© Blackwell Publishing Limited
1997; Darlington et al. 1998; Ishiura et al. 1998). How-
ever, the molecular events that cause these fundamental
feedback loops to oscillate stably with an approximate
periodicity of 24 h remain unclear. It has been postu-
lated in the general delay models that the single most
important parameter driving this periodicity is a ‘time-
delay’ event that regulates the activity and /or localization
of the core clock genes/proteins (Scheper et al. 1999;
Lema et al. 2000). For example, in Drosophila, PER and
TIM first undergo cytoplasmic accumulation, after which
the PER:TIM complexes enter the nucleus.This sequence
of temporally spaced events is believed to provide the
time-delay between the transcription of PER and TIM
and the inhibition of CLK:CYC transcriptional activity
by the PER:TIM complex. (Edery et al. 1994; Seaz &
Genes to Cells (2003) 8, 973–983 973
 T Tamaru et al.

Figure 1 Characterization of the anti-mBMAL1 antibody. (A) The anti-mBMAL1 antibody was used in immunoblot analysis of (a) the
crude lysate (10 µg protein) and anti-Flag immunoprecipitates of Cos7 cells transfected with pCMV-tag2B (Mock) or pCMV-tag2B/
Flag-mBMAL1b and (b, c) of the crude lysate (10 µg protein) of mouse brains and anti-BMAL1 immunoprecipitates prepared from
200 µg of brain protein. +Antigen, the signal was absorbed with the GST-BMAL1 protein to which the antibody was raised (20 µg protein/
mL). +λPPase, an aliquot of the immunoprecipitates was treated with λ protein phosphatase. (B) NIH-3T3 fibroblasts were stained using
the anti-mBMAL1 antibody. +Antigen, the signal was absorbed with the GST-BMAL1 protein to which the antibody was raised (20 µg
protein/mL).

Young 1996). However, while this may be the crucial
event in the fly clocks, mammalian clocks appear to
involve other time delay events, since it has been
reported that in mammalian clocks, the time lags
between Per1/Per2 transcription and PER1/PER2 pro-
tein accumulation in the nucleus are smaller (∼4 h, 6–
9 h and ∼6 h in rat-1 fibroblasts, mouse liver and SCN,
respectively). Moreover, it does not appear that there is
an significant accumulation of cytoplasmic PER1/
PER2 before the nuclear entry during the mammalian
circadian cycle (Hastings et al. 1999; Lee et al. 2001;
Yagita et al. 2001a,b).
BMAL1 (MOP3) is a basic helix-loop-helix-PER-
ARNT-SIM (bHLH-PAS) protein that forms a hetero-
dimer with CLOCK and thereby acts as the positive
feedback driver for the transcription from the E box of
circadian-responsive genes (Gekakis et al. 1998). BMAL1
also behaves as a negative feedback regulator for its own
gene expression through hypothetical interlocked feed-
back loops (Yu et al. 2002). BMAL1 mRNA and protein
levels in the SCN and other peripheral clock cells oscil-
late robustly in a circadian manner (Abe et al. 1998; Oishi
et al. 2000; Tamaru et al. 2000; Yagita et al. 2001a,b). As
Mop3 (Bmal1) (−/−) mice immediately display com-
plete arrhythmic wheel-running activity in constant
darkness, it appears that BMAL1 is an essential and non-
redundant component in the mammalian clock (Bunger
et al. 2000). In addition, lower levels of Bmal1 transcripts
are found in all mutant mice with arrhythmic clocks,
including the Clock mutant, the Period2Brdm1 mutant and
the Cry1-Cry2 (−/−) mutant (mCRY-deficient mice)
(Oishi et al. 2000; Shearman et al. 2000). That BMAL1
is a crucial component of the molecular clockwork
suggests that the regulatory events that control its local-
ization and /or activation may serve as the crucial time-
delay events that drive the stable 24-h periodicity of
the circadian clock. To test this notion, in this paper, we
examined the temporal regulation of the subcellular
localization and phosphorylation of the BMAL1 protein
in serum-shocked NIH-3T3 fibroblasts, which are a
cellular model of circadian clocks (Akashi & Nishida
2000; Balsalobre et al. 1998).
Results
Characterization of the anti-mBMAL1 antibody
We generated a highly sensitive polyclonal antibody by
immunizing rabbits with recombinant GST-mBMAL1
N-terminal protein. The specificity of the affinity-
purified anti-mBMAL1 antibody was characterized as
follows. Immunoblot analysis revealed that the antibody
reacts with Flag-mBMAL1 in Flag-immunoprecipitates
of transfected COS7 cell lysates (Fig. 1A(a)). Immuno-
blot analysis of anti-BMAL1 immunoprecipitates of the
mouse brain also showed that the antibody recognized a
broad band spanning 70– 80 kDa and that this recognition
could be eliminated by adding the antigen to which the
antibody was raised (Fig. 1A(b)). When the anti-BMAL1
immunoprecipitates of the mouse brain were treated
with λ protein phosphatase, the broad band shifted into
a narrower 70 kDa band (Fig. 1A(c)). Thus, it appears

974 Genes to Cells (2003) 8, 973–983 © Blackwell Publishing Limited


Figure 2 Phosphorylation of the BMAL1 protein in NIH-3T3
fibroblasts. (A) Anti-BMAL1 immunoprecipitates of serum-shocked
NIH-3T3 fibroblasts lysed 30 h after the serum shock (200 µg
protein) were subjected to immunoblot analysis using the anti-
mBMAL1 antibody. +λPPase, aliquots of the immunoprecipitates
were treated with λ protein phosphatase. The phosphorylated
(p-BMAL1) and unphosphorylated (arrow) forms of BMAL1 are
indicated. (B) Immunocytostaining of serum-shocked NIH-3T3
fibroblasts 30 h after the serum shock with the anti-mBMAL1
antibody.
that the BMAL1 protein in the mouse brain is highly
phosphorylated. When we performed immunocyto-
staining of NIH-3T3 fibroblasts with the anti-mBMAL1
antibody, clear nuclear-positive signals that were specifi-
cally absorbed with the antigen were observed (Fig. 1B).
BMAL1 nucleocytoplasmic localization and
phosphorylation in NIH-3T3 fibroblasts show a
circadian pattern
We investigated whether the nucleocytoplasmic localiza-
tion and phosphorylation of BMAL1 follows a circadian
rhythm by using serum-shocked NIH-3T3 fibroblasts
as a model of circadian clock cells. Thus, we treated the
cells with 50% serum for 2 h and then 30 h later sepa-
rated their lysates into nuclear and cytosolic fractions.
Each fraction was then immunoprecipitated with the
Figure 3 Alteration of the levels of
BMAL1 and other clock-related proteins
in NIH-3T3 fibroblasts after serum shock.
NIH-3T3 fibroblasts were treated with
50% serum for 2h and then the nuclear and
cytosolic fractions were harvested at the
indicated times and subjected to immuno-
blot analysis using antibodies specific for
mBMAL1, CLOCK, CRY1, CRY2,
CKIe, active MAPK (pERK), ERK1/2
(ERK) and phospho-CREB (pCREB).
The phosphorylated (dotted line) and un-
phosphorylated (arrow) forms of BMAL1
are indicated.
© Blackwell Publishing Limited
Circadian BMAL1 nucleocytoplasmic shuttling
anti-mBMAL1 antibody and the immunoprecipitates
were subjected to immunoblotting with the same anti-
body.The broad 70– 80 kDa band was detected with both
fractions (Fig. 2A). λ protein phosphatase treatment
caused this band to shift and narrow into a 70 kDa band
(Fig. 2A), which indicates that the proteins in the upper
parts of the broad 70– 80 kDa signal are highly phosphor-
ylated BMAL1 proteins. When the serum-shocked NIH-
3T3 cells were subjected to immunocytostaining with
the anti-BMAL1 antibody, staining in the cytoplasm and
the nucleus was also observed (Fig. 2B), which is consistent
with the signals detected in the immunoprecipitates.
We then used immunoblot analysis to assess the changes
in the protein levels of BMAL1 and other clock-related
proteins in the nuclear and cytosolic fractions of serum-
shocked NIH-3T3 fibroblasts over the 48 h after serum
shock (Fig. 3). Before serum shock, considerable amounts
of BMAL1 were detected in the nuclear and cytosolic
fractions. However, after serum shock, the total and
phosphorylated BMAL1 levels in the nuclear fraction
showed robust circadian oscillatory patterns that peaked
at 24 h in the first cycle after serum shock. This pattern
was repeated in the second cycle (these data are all
plotted in Fig. 5A). The proportion of phosphorylated
BMAL1 in the nucleus increased progressively until
the peaking time (∼24 h) (Fig. 3). Interestingly, in the
cytosolic fraction, the total and phosphorylated BMAL1
levels showed a different pattern. First, the total cytoplasmic
levels peaked much earlier at ∼16 h (plotted in Fig. 5A).
Second, the ratios between the hyperphosphorylated
and unphosphorylated forms in the cytosol are almost
constant.
The nuclear levels of CLOCK changed in a similar
circadian pattern to that of nuclear BMAL1 but in
contrast showed unaltered high constitutive expression in
the cytosol (Fig. 3). We observed that in the nucleus, the
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 T Tamaru et al.
anti-CLOCK antibody recognizes a band that includes
heavier proteins as well as the predicted ∼100 kDa CLOCK
protein (Fig. 3).These may be the phosphorylated forms
of the CLOCK protein, as has been proposed earlier
(Lee et al. 2001). However, unlike nuclear BMAL1, the
phosphorylated CLOCK levels in the nucleus did
not change progressively during circadian cycle (Fig. 3).
Moreover, cytosolic CLOCK appeared to be in the
unphosphorylated form and showed high constitutive
expression. With regard to nuclear CRY1 and CRY2,
both proteins exhibited a slightly time-delayed circadian
pattern as compared with the profile of nuclear BMAL1
(Fig. 3).
Since CKIε and MAP kinases (ERKs) are known to
phosphorylate BMAL1 in vitro (Sanada et al. 2002; Eide
et al. 2002), we also examined the circadian profiles of
these kinases. The nuclear profile of CKIε changed in a
976 Genes to Cells (2003) 8, 973–983
Figure 4 Circadian nucleocytoplasmic
localization of BMAL1 in NIH-3T3
fibroblasts after serum shock. At various
time points after serum shock, NIH-3T3
fibroblasts were immunostained with the
anti-mBMAL1 antibody. The antibody
binding was detected using Alexa Fluor
488-labelled goat anti-rabbit IgG (Green)
as a secondary antibody. The cells were
also stained with DAPI to reveal the
nucleus (Blue).
similar circadian pattern to that of CRY1 and CRY2.
Thus, nuclear CKIε levels do not follow the same timing
as the progressive phosphorylation of nuclear BMAL1.
The activated ERK protein levels showed a similar cir-
cadian pattern to the profile of phosphorylated CREB
(Fig. 3), a transcription factor that is a nuclear target of
the MAP kinase pathway and that has been implicated
in circadian clock and photic entrainment (Gau et al.
1996).The nuclear and cytosolic levels of activated ERKs
showed only weak changes during the circadian cycle.
Relative to the first cycle, the circadian patterns of the
protein levels of activated and nuclear-translocated ERKs
and phosphorylated CREB became attenuated in the
second cycle. Thus, the nuclear and cytosolic levels of
activated ERKs and phosphorylated CREB do not follow
the same timing as the progressive phosphorylation of
nuclear BMAL1.
© Blackwell Publishing Limited
 Circadian BMAL1 nucleocytoplasmic shuttling

Figure 5 Time-delayed accumulation of

BMAL1 in the nucleus in NIH-3T3
fibroblasts after serum shock. Total RNA
harvested from NIH-3T3 fibroblasts at
various time points after serum-shock was
subjected to fluorescence-based real-time
PCR technology (TaqMan Real-Time
PCR) to quantify Bmal1 (A) and Per1 (B
(a)) mRNA.The reactions were performed
with 40 ng total RNA/reaction. Bmal1
(A) and Per1 mRNA (B (a)) mRNA values
from triplicate experiments are shown as
means ± SEM (Blue rectangle). The levels
of BMAL1 protein in the immunoblot
shown in Fig. 3 were also quantified by
densitometry of the anti-BMAL1 positive
signal in the immunoblot using a com-
puterized image analyzer (A). The values
from triplicate experiments are shown as
means ± SEM (Nuclear; Green circle,
Cytosolic; Red triangle). Staining with
anti-PER1 antibody (Green) and DAPI
(Blue) are shown (B (b)). The values of
nuclear PER1-positive cells (%) from
triplicate experiments are plotted as means
± SEM (B (a)).

BMAL1 levels decreased rapidly while at the same time

BMAL1 nuclear accumulation shows a 12–16 h time
lag after Bmal1 transcription
To further characterize the temporal relationships between
the transcription, nucleocytoplasmic shuttling and phos-
phorylation of BMAL1 in serum-shocked NIH-3T3
fibroblasts, we examined Bmal1 transcription by quanti-
tative PCR and BMAL1 nucleocytoplasmic localization
by immunocytochemistry. Bmal1 mRNA levels oscillated
with a peak at 8 h after the serum shock in the first cycle
(Fig. 5A). As the mRNA levels decreased, BMAL1 protein
progressively accumulated in the cytoplasm (Figs 3, 4, 5A;
12, 13, 14, 15 and 16 h after serum shock).With regard to
BMAL1 protein in the nucleus, these levels also began to
increase progressively and peaked 12–16 h (at the 1st and
2nd cycles) after the Bmal1 mRNA levels peaked (Figs 3,4
and 5A; ∼24 h after serum shock).Thereafter, the nuclear
the total and BMAL1 levels in the cytoplasm increased
(Figs 3, 4, 5A; 28, 29, 30, 31 and 32 h after serum-shock).
In contrast to BMAL1, PER1 nuclear accumulation
in serum-shocked fibroblasts shows only a ∼4 h time lag
after Per1 transcription. Per1 mRNA levels oscillated
with a peak at 28 h after the serum shock (Fig. 5B(a))
and nuclear PER1 levels showed a peak at 32 h during
the circadian cycle without any evidence of slow accu-
mulation in the cytoplasm (Fig. 5B(a,b)).
The circadian subcellular localization and
phosphorylation of BMAL1 are blunted in
CRY-deficient MEFs
We examined whether the nucleocytoplasmic localization
and phosphorylation of BMAL1 also follows a circadian

© Blackwell Publishing Limited Genes to Cells (2003) 8, 973–983 977

 T Tamaru et al.
rhythm in serum-shocked mCry1/mCry2-deficient mouse
embryonic fibroblasts (CRY-deficient MEFs), which
lack a functional biological clock (Yagita et al. 2001a,b).
Previous studies of CRY-deficient MEFs have shown
that the RNA levels of BMAL1 are arrhythmic and are
maintained at moderate levels during the circadian cycle.
We found that cytoplasmic and nuclear BMAL1 protein
levels were also arrhythmic and maintained at moderate
levels (Fig. 6A). Moreover, unlike what is observed in the
wild-type MEFs, circadian subcellular BMAL1 localiza-
tion changes was not observed in CRY-deficient MEFs,
even at unicellular levels, as revealed by immunocyto-
chemistry that showed staining in nuclei and cytoplasms
at moderate intensities (Fig. 6B). Supporting these
observations is that it has been previously reported that
the cytoplasmic and nuclear BMAL1 levels in the liver of
the CRY-deficient mouse are also arrhythmic and are
maintained at substantially reduced levels (Lee et al. 2001).
In addition, in the CRY-deficient MEFs, the cytoplasmic
and nuclear BMAL1 proteins were constantly detected as
978 Genes to Cells (2003) 8, 973–983
Figure 6 The circadian phosphorylation
and nucleocytoplasmic localization of
BMAL1 are blunted in CRY-deficient MEFs.
(A) Wild type [W] and CRY-deficient
MEFs [(−/−)] were treated with 50% serum
and their nuclear and cytosolic fractions
were obtained at the indicated times there-
after and subjected to immunoblot analysis
using antibodies specific for mBMAL1,
CLOCK, CRY1, CRY2, CKIε, active
MAPK (pERK) and ERK1/2 (ERK). The
phosphorylated (dotted line) and unphos-
phorylated (arrow) forms of BMAL1 are
indicated. (B) At various time points after
serum shock, wild type [Wild] and CRY-
deficient MEFs [Cry (−/−)] were immuno-
stained with the anti-mBMAL1 antibody.
hyperphosphorylated forms (Fig. 6A). Again, this is sup-
ported by previous observations of the liver of the CRY-
deficient mouse, as hyperphosphorylated BMAL1 is
detected in the nuclear fraction in the liver, although not
in the cytoplasm (Lee et al. 2001).
The nuclear levels of CLOCK in CRY-deficient MEFs
were at substantially higher levels, which is due to increase
of the unphosphorylated forms (Fig. 6A). Cytosolic acti-
vated ERKs showed lower levels in CRY-deficient MEFs
before serum shock but slightly higher levels after serum
shock (Fig. 6A). Nuclear total ERKs and nuclear/cytosolic
CKIε proteins were at higher levels in CRY-deficient
MEFs, but the activated ERKs did not differ significantly
between the wild-type and CRY-deficient MEFs (Fig. 6A).
The circadian nuclear export of BMAL1 is inhibited
with Leptomycin B
To demonstrate that the nucleocytoplasmic shuttling of
BMAL1 in the cultured fibroblasts is controlled by the
© Blackwell Publishing Limited

Figure 7 Effect of Leptomycin B on the circadian
nucleocytoplasmic localization of BMAL1:CLOCK in cultured
fibroblasts. After adding cycloheximide (50 µg/mL; Calbiochem-
Novabiochem) or methanol (as a control) 18hr after serum shock,
NIH-3T3 fibroblasts (A), wild type [Wild] and CRY-deficient
MEFs [Cry (−/−)] (B) were treated with Leptomycin B (LMB)
(10 ng/mL; Calbiochem-Novabiochem) or methanol (Control)
from 24–30 hr after serum shock. Samples of the fibroblasts were
analysed with immunocytochemistry (A (a, b), B) or immuno-
blotting (A (c); nuclear [N] and cytosolic [C] fractions) using the
anti-mBMAL1 and anti-CLOCK antibody. Staining with anti-
BMAL1 antibody (Green) and DAPI (Blue) are shown (A (a), B).
The values of nuclear BMAL1-dominant cells (%) in NIH-3T3
cells from triplicate experiments are plotted as means ± SEM (A
(b)). (***P < 0.001).
circadian clock, we examined the effect of Leptomycin
B, a potent inhibitor of CRM-1-mediated nucleoex-
port, on the nucleocytoplasmic shuttling of BMAL1. To
eliminate de novo synthesized BMAL1 protein, the cells
were pretreated with cycloheximide for 6 h (18–24 h
after serum shock). Treatment with Leptomycin B dur-
ing the 24– 30 h after serum shock, when the circadian
© Blackwell Publishing Limited
Circadian BMAL1 nucleocytoplasmic shuttling
nuclear export of BMAL1 was likely to be occurring,
dramatically increased the dominant nuclear BMAL1
signals in NIH-3T3 cells (Fig. 7A(a–c)) and wild-type
MEFs but not in CRY-deficient MEFs (Fig. 7B).Without
cycloheximide, BMAL1 also accumulated in the nucleus
after Leptomycin B treatment (Fig. 7A(c)). CLOCK
protein also accumulated in the nucleus after Leptomycin
B treatment with a similar pattern to BMAL1. These
data clearly demonstrate that BMAL1 and CLOCK are
exported from the nucleus via the CRM-1-mediated
pathway in a circadian clock-dependent manner.We also
addressed the effect of cycloheximide on the expression
pattern of BMAL1. Only the highly phosphorylated forms
of BMAL1 remained in cycloheximide-treated cells
(Fig. 7A(c)).
Discussion
We found that in cells with a functional clock, BMAL1
undergoes progressive cytoplasmic accumulation followed
by a much later nuclear entry. Thus, in serum-shocked
NIH 3T3 cells and wild-type MEFs, we found that
BMAL1 protein progressively accumulates in the cyto-
plasm with peaks observed at 12–16 h after serum shock.
This is followed by a diminishment in the cytoplasmic
protein levels and a concomitant increase of nuclear
BMAL1 protein with a peak at ∼24 h. Thereafter, the
nuclear BMAL1 levels exhibit a dramatic reduction at
28– 32 h. This is due in part to nuclear export since at
this time point, the cytoplasmic BMAL1 levels start to
increase again while Bmal1 gene expression in the second
cycle has not yet begun. Strongly supporting this notion
is that this increase of cytoplasmic BMAL1 is dramati-
cally inhibited by the nuclear export inhibitor Leptomycin
B. Proteolysis may also contribute to the rapid decline
of nuclear BMAL1 because cycloheximide-treated cells
that lack de novo protein synthesis showed significantly
lower nuclear levels of BMAL1. It may be that certain
temporally specific dephosphorylation events may con-
tribute to the destabilization of BMAL1 because only the
highly phosphorylated forms of BMAL1 remain in both
the nucleus and the cytoplasm at this time point. There-
after, cytoplasmic BMAL1 levels reach a second peak at
∼32 h.
These observations indicate that the BMAL1 protein
undergoes temporally regulated subcellular localization
in mammalian cells that have a functional circadian clock.
This temporal distance between transcription and nuclear
localization is larger than has been reported previously
for any other mammalian clock protein. The temporal
regulation of BMAL1 localization is supported by the
fact that the subcellular localization of BMAL1 protein
Genes to Cells (2003) 8, 973–983 979
 T Tamaru et al.
Figure 8 Nuclear-dominant localization of BMAL1 in the
mouse liver throughout the circadian cycle. (A) The nuclear and
cytosolic fractions of mouse livers harvested at the indicated
circadian time points were subjected to immunoblot analysis using
anti-mBMAL1 antibody. The phosphorylated (dotted line) and
unphosphorylated (arrow) forms of BMAL1 are indicated. (B) At
circadian time 10 and 22, the BMAL1 in the mouse liver was
immunohistochemically detected with the anti-mBMAL1 anti-
body. The antibody binding was detected using Alexa Fluor 488-
labelled goat anti-rabbit IgG as a secondary antibody. The cells
were also stained with PI to reveal the nucleus.
in CRY-deficient MEFs, which lack a functional circa-
dian clock, do not change over time. A recent report
has suggested that in the mouse liver, BMAL1 does
not accumulate significantly in the cytoplasm before
the nuclear entry (Lee et al. 2001). We also observed this
when we subjected mouse liver to immunohistochemical
and immunoblot analysis (Fig. 8). This difference sug-
gests that BMAL1 may be subjected to different post-
translational regulatory mechanisms in the molecular
clockworks of different cell types. Nevertheless, another
recent report (Yagita et al. 2001a,b) has suggested that
cultured fibroblasts exhibit similar circadian gene expres-
sion profiles to the SCN. This supports the importance
of cultured fibroblasts as a model for the central circadian
clock. Moreover, according to a recent report that was
published during the preparation of this paper, it suggests
that the similar post-translational regulatory mechanisms
that control subcellular localization of CLOCK, which
is shown to undergo BMAL1-dependent nuclear entry,
in fibroblasts may also occur in the SCN (Kondratov
et al. 2003). However, further analyses will be required
to adequately define the circadian regulatory mecha-
nisms that drive the subcellular localization of BMAL1 in
different cell types, especially neurones in the SCN.
Our immunocytochemical analysis shows that in cells
with a functional clock, BMAL1 slowly accumulates in
the cytoplasm before its nuclear entry.These data consti-
tute the first immunocytochemical evidence for the
980 Genes to Cells (2003) 8, 973–983
temporal regulation of endogenous BMAL1 protein
localization.We also demonstrated that the nucleoexport
of BMAL1 in these cells involves the CRM-1-mediated
pathway In CRY-deficient MEFs that lack a functional
biological clock, the nucleoexport-blocking effect of
Leptomycin B was not observed. This suggests that
nuclear export of BMAL1 is largely dependent on the
circadian clock. It may be that circadian time-specific
BMAL1 phosphorylation events may be required before
its CRM-mediated nucleoexport can take place. These
observations of the nucleocytoplasmic shuttling of BMAL1
are supported by the presence in the mBMAL1 protein
of several well-conserved motifs that are involved in
nucleocytoplasmic localization (Seaz & Young 1996;
Yagita et al. 2001a,b), namely, two well-conserved nuclear
localization signals (NLS) in the N-terminal region
(N36RKRK) and the bHLH region (K82RRRR), two
well-conserved nucleoexport signals (NES) in the PAS B
region (L311SCLVAIGRL and I360LAYLPQELL), and a
partially conserved cytoplasmic localization domain (CLD)
in amino acids 398– 460.
Nuclear BMAL1 protein in serum-shocked fibroblasts
was gradually phosphorylated and dephosphorylated in
a temporal manner, but in contrast cytoplasmic BMAL1
protein exhibited the same phosphorylation pattern
throughout the circadian cycle. This indicates that the
phosphorylation of nuclear and cytoplasmic BMAL1
proteins may be differently regulated and that the phos-
phorylation of nuclear BMAL1 protein may be regulated
by circadian clock mechanisms. Supporting this notion
is that in CRY-deficient MEFs, which lack a functional
biological clock, both nuclear and cytoplasmic BMAL1
proteins consisted of moderate levels of hyperphosphory-
lated forms throughout the entire circadian cycle. As
reported recently, BMAL1 is also temporally phosphory-
lated in the mouse liver (Lee et al. 2001). However, the
circadian profile of BMAL1 phosphorylation in the liver
is different from that in NIH-3T3 cells. Moreover, in
contrast to wild-type and CRY-deficient fibroblasts,
cytoplasmic BMAL1 in the liver of both wild-type and
CRY-deficient mice do not show hyperphosphorylated
forms. It may be that the BMAL1 protein in different
cells is subjected to different phosphorylation events.
Further analyses will be required to adequately define
the circadian phosphorylation of BMAL1 in different
cell types, especially SCN neurones.
cBMAL1 can be phosphorylated in vitro by ERK at
Ser-527, Thr-534 and Ser-599, which are largely con-
served in mBMAL1 (Sanada et al. 2002). Moreover,
CKIε can also phosphorylate BMAL1 in vitro (Eide et al.
2002) and we found a variety of predicted Ser/Thr
phosphorylation motifs in BMAL1. However, we found
© Blackwell Publishing Limited

in this paper that the temporal changes of nuclear ERKs
and CKIε do not significantly match the timing of the
progressive phosphorylation of nuclear BMAL1. These
data do not necessarily exclude the possibility that ERKs
and CKIε phosphorylate BMAL1 in vivo, but they do
suggest that other kinases or phosphatases may be
responsible for the circadian phosphorylation states of
BMAL1. However, in CRY-deficient cells, higher levels
of nuclear and cytoplasmic CKIε protein are observed
along with hyperphosphorylation of the nuclear and
cytoplasmic BMAL1 proteins in these cells.This suggests
that CKIε may be involved in these hyperphosphoryla-
tion events in CRY-deficient cells. Notably, in the
mouse liver (Lee et al. 2001), the lack of nuclear accu-
mulation of CKIε correlates with the lack of nuclear
PER1/PER2. As reported previously, in CRY-deficient
MEFs, PER1 protein can enter the nucleus (Yagita et al.
2000). These observations together suggest that CKIε
may also be involved in the nuclear localization of PER1.
Thus, CKIε may play different roles in the nucleocyto-
plasmic localization of clock proteins in different cell
types.
Unlike CKIε, the levels of activated ERKs are not sig-
nificantly elevated in CRY-deficient cells. This suggests
that these kinases are not responsible for the constantly
hyperphosphorylated nuclear and cytoplasmic BMAL1
proteins in these cells.
The changing levels of nuclear CLOCK protein over
circadian time matched the circadian pattern of nuclear
BMAL1 levels. This suggests that the subcellular locali-
zation of BMAL1 affects the localization of CLOCK.
Supporting this is that the subcellular localization of
CLOCK in CRY-deficient MEFs does not change
throughout the entire experimental period. Moreover,
according to a recent report that was published during
the preparation of this paper, the circadian clock-
controlled nuclear entry of CLOCK is impaired in
BMAL1-deficient MEFs, and hyperphosphorylation of
BMAL1 is dependent on its interaction with CLOCK
(Kondratov et al. 2003). We demonstrated in our paper
here that BMAL1 and CLOCK undergo clock-controlled
nucleoexport via the CRM-1-mediated pathway. From
these findings, we propose that the nucleocytoplasmic
shuttling and interaction of BMAL1:CLOCK is controlled
by circadian clock-dependent post-transcriptional events
such as temporal phosphorylation.
The timing of the nuclear accumulation of BMAL1
and CLOCK fits well with the expression of the clock-
responsive genes Per1, which are the targets of the
BMAL1:CLOCK complex, as the expression of these
genes peaks 20–24 h after serum shock (Akashi & Nishida
2000). Moreover, the nuclear accumulation of CRY1/
© Blackwell Publishing Limited
Circadian BMAL1 nucleocytoplasmic shuttling
CRY2, the major negative regulator of BMAL1:CLOCK-
driven gene expression, is slightly delayed compared to
the timing of the nuclear accumulation of BMAL1
and CLOCK and peaks when Per1 gene expression is
repressed (Akashi & Nishida 2000). These observations
strongly suggest that the BMAL1:CLOCK directly drives
the circadian expression of Per1 in NIH-3T3 cells that
have been stimulated to enter a circadian rhythm. It is
also possible that nuclear phosphorylated BMAL1 plays
an important role in the transcriptional activation by
BMAL1:CLOCK because the phosphorylation pattern
of nuclear BMAL1 also matches the transcriptional
pattern of Per1. Supporting this notion is that a repor-
ter assay employing the Per1-promoter revealed that
BMAL1 phosphorylation by MAP kinase and CKIε
affects BMAL1:CLOCK-mediated transcriptional activity
(Eide et al. 2002; Sanada et al. 2002).
In summary, our observations suggest that the subcel-
lular localization and phosphorylation states of BMAL1,
a positive limb in the circadian core feedback loops
involved in the transcription from the E box, are impor-
tant events in the stably oscillating molecular clockwork
in cultured fibroblasts, as well as in negatively regulating
limbs such as PER and CRY. Supporting this notion is
that the temporal Per1 transcription pattern matches the
timing of the time-delayed nuclear entry, accumulation
and phosphorylation of BMAL1, which suggests that
BMAL1 acts as a positive driver in the molecular circa-
dian clockwork.
Experimental procedures
Preparation of antibodies
Glutathione S-transferase (GST)-mBMAL1 N-terminal (amino
acids 1–73; this peptide shows no homology to BMAL2) (Hogenesch
et al. 2000) and GST-mCRY2 C-terminal (amino acids 524–592)
antigens were produced and then used to immunize rabbits as
previously described (Tamaru et al. 2000). After removing possible
GST-recognizing antibodies by using Hitrap-NHS conjugated
with GST, the antisera were subjected to affinity purification using
Hitrap-NHS conjugated with the antigen. Both the anti-mBMAL1
antibody and the anti-mCRY2 antibody specifically recognize
their target proteins in immunochemical analysis (Fig. 1 and data
not shown).
Cell culture, preparation of tissue samples,
immunoprecipitation and immunoblot analysis
Cell culture and the serum-shocking of NIH-3T3 fibroblasts were
performed according to protocols described by Akashi and
Nishida (Akashi & Nishida 2000). Flag-mBMAL1 was over-
expressed by transfecting COS7 cells with mBMAL1b cDNA
Genes to Cells (2003) 8, 973–983 981
 T Tamaru et al.
ligated into pCMV-Tag2B (Stratagene Cloning Systems, La Jolla,
CA, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad,
USA). The mouse brain and liver samples were prepared from
male 8-week-old C57/BL6 mice (Sankyo Laboratory Service,
Tokyo, Japan). Nuclear and cytosolic fractions at the indicated
time points were prepared as previously described (Tamaru et al.
2000). For immunoprecipitation analysis, the samples were
incubated at 4 °C for 16 h with 1 µg of antibody in TTBS
(Tris-buffered saline; TBS with 0.1% Tween-20) containing Ser/
Thr phosphatase inhibitor cocktail (Sigma-Aldrich, Tokyo, Japan)
and complete™ protease inhibitor cocktail (Roche Diagnostics,
Tokyo, Japan). After incubating with Protein G-Sepharose
(Amersham Pharmacia Biotech, Tokyo, Japan), the beads were
washed with TTBS. The immunoprecipitates were treated with
l protein phosphatase (500 units) (Calbiochem-Novabiochem,
Darmstadt, Germany) at 30 °C for 2 h. Immunoblot analyses
were performed as previously described (Tamaru et al. 2000).
The following antibodies were used: anti-active MAPK antibody
(Promega, Tokyo, Japan), anti-CLOCK antibody (Calbiochem-
Novabiochem), anti-CKIe antibody (Takano et al. 2000), anti-
ERK1/2 and anti-phospho CREB antibodies (Upstate, Lakeplacid,
USA), anti-PER1 antibody (kindly donated by Dr Okamura;
Yagita et al. 2001a,b) and anti-CRY1 antibody (Alpha Diagnostic,
San Antonio, TX, USA).
Immunocytochemistry
At the indicated time points, the cells cultured on slide glasses were
fixed with 4% paraformaldehyde in TBS at room temperature for
30 min. After a brief incubation with 0.3% Triton X-100 in TBS,
blocking was performed with BLK buffer (TBS with 0.5% BSA
and 0.1% Triton X-100) at room temperature for 1 h. The speci-
mens were incubated with the anti-mBMAL1 antibody (20 µg/
mL in BLK) at 4 °C for 48 h. The antibody binding was detected
by using Alexa Fluor 488-labelled goat anti-rabbit IgG as a sec-
ondary antibody, while nuclear staining was performed by using
DAPI (Molecular Probes, Eugene, USA). The slides were then
visualized by laser confocal microscopy (Bio-Rad Laboratory,
Tokyo, Japan).
Immunohistochemistry
Mice were deeply anaesthetized under a dim red light and per-
fused intracardially with ice cold saline and then with 4% parafor-
maldehyde (Sigma, St Louis, MO, USA) in phosphate-buffered
saline (PBS). Livers were removed, post-fixed in 4% PFA in PBS
for 2 h at room temperature, and cryoprotected in 20% sucrose for
two nights. The brains were sliced using a cryomicrotome
(CM1900, Leica, Germany) into 10 µm-thick sections. Immuno-
histochemical analyses of BMAL1 were performed by using anti-
mBMAL1 antibody (20 µg/mL) as the primary antibody. The
antibody binding was detected by using Alexa Fluor 488-labelled
goat anti-rabbit IgG as a secondary antibody, while nuclear stain-
ing was performed by using PI (Molecular Probes) according to
the manufacturer’s instruction.This was followed by laser confocal
microscopy (Bio-Rad).
982 Genes to Cells (2003) 8, 973–983
Quantitative PCR
At the indicated time points, total RNA was extracted using an
RNAwith™ and DNA-free kit (Ambion, Austin, TX, USA).
RNA quantification was performed using fluorescence-based
real-time PCR technology (TaqMan Real-Time PCR, Applied
Biosystems, Tokyo, Japan) as previously described (Tamaru et al.
2003) using the following probes (5′-labelled with FAM and 3′-
labelled with TAMRA) and primers: mBMAL1-For, GAC CTT
ATT GGC CAG AGC TTG TT; mBMAL1-Rev, CGC AGT
GTC CGA GGA AGA TAG; mBMAL1-Probe, CCT GCA TCC
AAA AGA TAT TGC CAA AGT TAA GGA; the probes and
primers for mPER1 were previously described (Tamaru et al.
2003). As a control, GAPDH mRNA was amplified from the
same RNA samples. The relative mRNA abundance was calcu-
lated using the comparative delta-Ct method according to the
manufacturer’s protocol.
Acknowledgements
The authors thank Drs Hitoshi Okamura, Masaaki Ikeda and
Steven M. Reppert for kindly donating the anti-PER1 antibody,
the mBMAL1 cDNA, and the mCRY1/mCRY2 cDNA, respec-
tively. This work was supported by a grant-in-aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science
and Technology of Japan (to T. Tamaru and K. Takamatsu), the
Scientific Research Promotion Fund from the Japan Private School
Promotion Foundation (to K.Takamatsu), and the Project Research
Grant from Toho University School of Medicine (to T.Tamaru).
This work was performed within the Cooperative Research Pro-
gram of the Institute for Protein Research, Osaka University.
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Received: 22 July 2003
Accepted: 18 September 2003
Genes to Cells (2003) 8, 973–983 983