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The Journal of Neuroscience, February 1, 2000, 20(3):986-991
Role of Circadian Activation of Mitogen-Activated Protein Kinase
in Chick Pineal Clock Oscillation
Kamon
Sanada,
Yuichiro
Hayashi,
Yuko
Harada,
Toshiyuki
Okano, and
Yoshitaka
Fukada
Department of Biophysics and Biochemistry, Graduate School of
Science, The University of Tokyo and CREST, Japan Science and
Technology Corporation, Tokyo 113-0033, Japan
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ABSTRACT |
A circadian pacemaker generates a rhythm with a period of ~24 hr
even in the absence of environmental time cues. Several photosensitive neuronal tissues such as the retina and pineal gland contain the autonomous circadian pacemaker together with the photic-input pathway
responsible for entrainment of the pacemaker to the daily light/dark
cycle. We show here that, in constant darkness, chick pineal
mitogen-activated protein kinase (MAPK) exhibited an in vivo circadian rhythm in tyrosine phosphorylation and in
enzymatic activity with a peak during subjective night. Phosphorylated
and hence activated MAPK was rapidly dephosphorylated after light illumination during the nighttime when light induces a phase-shift of
the pacemaker. The circadian rhythmicity in MAPK phosphorylation was
also observed in the cultured pineal gland, and importantly, MAPK
kinase inhibitor treatment during subjective night not only shifted the
time-of-peak of MAPK phosphorylation but also induced a remarkable
phase-delay of the circadian pacemaker. These results indicate an
important role of MAPK for time keeping in circadian clock systems.
Key words:
circadian rhythm; pineal gland; tyrosine phosphorylation; mitogen-activated protein kinase; chick
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INTRODUCTION |
Many organisms, from bacteria to
humans, contain endogenous circadian pacemakers that control daily
rhythms in behavior and physiology (Pittendrigh, 1993 ). These rhythms
are synchronized (entrained) to a period of 24 hr by environmental
stimuli, most commonly by light, but they are sustained even under
constant conditions (Takahashi, 1995; Dunlap, 1999). The circadian
pacemaker seems to generally consist of positive/negative elements
forming a transcription/translation-based negative feedback loop
(Dunlap, 1999). The positive elements in the loop activate
transcription of negative element genes and hence increase their
protein levels (Crosthwaite et al., 1997; Darlington et al., 1998;
Gekakis et al., 1998). The negative elements then inhibit the positive
element-induced transactivation of their own promoters to decrease the
transcript levels (Darlington et al., 1998). Thus, the levels of
negative elements (and their mRNAs) exhibit a circadian oscillation.
This fundamental frame of negative feedback loop may be ubiquitous because the negative and positive elements have been identified in a
variety of organisms (Hardin et al., 1990; Aronson et al., 1994; Sehgal
et al., 1995; Crosthwaite et al., 1997; King et al., 1997; Allada et
al., 1998).
In addition to the transcriptional regulation of these clock genes,
various post-translational mechanisms such as protein phosphorylation,
nuclear entry, and proteolysis have been implicated in regulation of
the circadian rhythmicity in the clock protein levels and their
activities (Zeng et al., 1996; Garceau et al., 1997; Kloss et al.,
1998; Lee et al., 1998; Price et al., 1998). These post-translational
processes are likely to contribute to maintenance of period lengths of
the molecular oscillations by generating an appropriate time lag
(Dunlap, 1999). Especially, protein phosphorylation plays a critical
role in the circadian clock systems of various organisms (Roberts et
al., 1989; Comolli et al., 1994; Zeng et al., 1996; Garceau et al.,
1997; Lee et al., 1998), and an important contribution of PERIOD
protein phosphorylation is clearly demonstrated in
Drosophila (Kloss et al., 1998; Price et al., 1998). In
vertebrates, however, less is known about the roles of protein
phosphorylation in autonomous circadian pacemakers that are localized
in specific neuronal tissues such as the suprachiasmatic nucleus (SCN),
pineal gland, and retina (Deguchi, 1979; Kasal and Menaker, 1979; Green
and Gillette, 1982; Cahill and Besharse, 1993; Tosini and Menaker,
1996). Because tyrosine kinases are particularly abundant in vertebrate
neuronal tissues (van der Geer et al., 1994; Neet and Hunter, 1996), we
have focused our attention on an involvement of protein tyrosine
phosphorylation in the circadian pacemaker and light-input pathway.
Among the clock containing tissues, the chick pineal gland retains both the pacemaker and photoreceptive molecules such as pinopsin within a
single cell, and exhibits the circadian rhythm in melatonin production
(Deguchi, 1979; Takahashi et al., 1980; Zatz et al., 1988; Okano et
al., 1994; Nakahara et al., 1997). Thus, it represents a good organism
for the study of the circadian clock systems in vertebrates. Here we
report that chick pineal MAPK activity not only oscillates with a
circadian rhythm but also affects the endogenous pacemaker
oscillation, suggesting the participation of MAPK in the circadian
feedback loop.
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MATERIALS AND METHODS |
Preparation of chick pineal homogenate. Animals were
treated in accordance with the guidelines of University of Tokyo.
Pineal glands were isolated from seven chicks and homogenized (10 strokes) using Teflon homogenizer with 150 µl of ice-cold solution
(10 mM Tris-HCl, 1 mM
Na3VO4, 4 µg/ml
aprotinin, and 4 µg/ml leupeptin, pH 8.0). The homogenate was
supplemented with 1% (w/v) SDS (final concentration), boiled for 5 min, and then passed through a 26 gauge needle. Preparation of the
pineal homogenate was performed under dim red light (>640 nm).
Immunoprecipitation. Pineal homogenate (100 µl) was mixed
with 900 µl of ice-cold lysis buffer [20 mM Tris-HCl,
1% (v/v) Triton X-100, 10% (v/v) glycerol, 137 mM NaCl, 2 mM EDTA, 50 mM NaF, 1 mM
Na3VO4, 4 µg/ml
aprotinin, and 4 µg/ml leupeptin, pH 8.0]. The lysate was precleared
by incubation for 30 min with 50% slurry of protein G-Sepharose (50 µl), and then incubated for 8 hr at 4°C with 2.5 µg of
anti-PanERK antibody (Transduction Laboratories, Lexington, KY),
followed by incubation with 50% slurry of protein G-Sepharose (20 µl) for 4 hr at 4°C. The immunoprecipitates were washed three times
with the lysis buffer and subjected to immunoblotting.
Immunoblotting. Proteins separated by SDS-polyacrylamide
(13% for Fig. 1a or 8.5% for
others) gels were transferred to polyvinylidene difluoride membranes.
The blots were incubated at 37°C for 1 hr with 1% (w/v) skim milk
[or 3% (w/v) BSA for anti-phosphotyrosine antibody] in TBS (in
mM: 50 Tris-HCl, 140 NaCl, and 1 MgCl2, pH 7.4), and then incubated at 4°C
overnight with primary antibodies in the blocking solution.
Phosphotyrosine was detected by anti-phosphotyrosine antibody (1:1000
dilution; Transduction Laboratories). MAPK was detected by anti-PanERK
antibody (1:5000; Transduction Laboratories). Phosphorylated form of
MAPK was detected by anti-phospho-MAPK antibody (1:2000, New England
Biolabs, Beverly, MA). Tryptophan hydroxylase was detected by
anti-tryptophan hydroxylase antibody (1:400; Chemicon, Temecula, CA).
The blots were visualized by an enhanced chemiluminescence detection
system (Renaissance; NEN). When reprobed, the blots were stripped
according to manufacturer's protocol.
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Figure 1.
Tyrosine dephosphorylation of chick pineal MAPK
after exposure to light. Chicks were entrained to a 12 hr light/dark
cycle for 7 d and then transferred to constant darkness.
a, On day 8, the pineal glands were isolated from chicks
at CT 20 in the dark (lanes 1, 3) or just after exposure
to white light (200-300 lux) for 15 min (lanes 2, 4). The pineal homogenates were immunoblotted with
anti-phosphotyrosine antibody (lanes 1, 2), and the blot
was reprobed with anti-MAPK antibody (lanes 3, 4). b, The chicks were exposed to white
light (200-300 lux) at CT 18.0 on day 8 and then killed at CT 18.0, 18.3, 18.7, and 19.0 in the light. Control animals kept in the dark
were killed at CT 17.7 and CT 19.3. MAPK in each pineal homogenate was
immunoprecipitated, immunoblotted with anti-phosphotyrosine antibody
(top panel), and then reprobed with anti-MAPK
antibody (bottom panel).
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Immune complex kinase assay. MAPK was immunoprecipitated
with anti-ERK2 antibody as described (Cook and McCormick, 1993), and
MAPK activity in the immunoprecipitate was assayed at 30°C for 20 min
in the presence of 10 µM
[ -32P] ATP (160 µCi/ml) and 8 µg
of myelin basic protein (MBP) as substrates. Proteins in the mixtures
were resolved by SDS polyacrylamide (13%) gel, and the incorporation
of 32P into MBP was quantitated using an
image analyzer (FLA2000; Fujifilm, Tokyo, Japan).
Organ culture. One-day-old male chicks were entrained to a
12 hr light/dark cycle for at least 7 d. Chick pineal glands were cultured as described (Deguchi, 1979) with some modifications. In
brief, the pineal glands were isolated 1-2 hr before the end of the
light period, and then cultured on Millicell CM membranes (Millipore,
Bedford MA; four glands in a cell of 30 mm diameter) in 1.9 ml of
BGJb medium supplemented with 10% heat-inactivated fetal calf
serum, 100 µg/ml streptomycin sulfate, and 100 U/ml penicillin under
85% O2 and 5%
CO2 at 38°C. After the light period, the
culture plates were transferred to constant darkness. To assess the
phosphorylation state of MAPK, the cultured tissues (four pineal
glands) were collected at various circadian times, rinsed with ice-cold
TBS, and immediately homogenized (10 strokes) using Teflon homogenizer
with 150 µl of lysis buffer. The homogenate was then mixed with
SDS-PAGE sample solution, boiled for 5 min, and passed through a 26 gauge needle. Preparation of the tissue homogenate was performed under
infrared light (>800 nm) with the aid of darkroom goggles (NEC, Tokyo, Japan).
Immunofluorescence microscopy. Cultured pineal glands were
fixed with 4% (w/v) paraformaldehyde in PBS (in mM: 10
Na-phosphate, 140 NaCl, and 1 MgCl2, pH 7.4) for
1 hr at 4°C. The frozen sections (10 µm thickness) were incubated
with 3% (w/v) BSA in PBS for 1 hr at room temperature, and then
incubated with anti-phospho-MAPK antibody (1:50; New England Biolabs)
in the blocking solution for 18 hr at 4°C, followed by incubation
with fluorescein-conjugated goat anti-rabbit IgG (1:500; Vector
Laboratories, Burlingame, CA) in the blocking solution for 1 hr at room
temperature. The sections were mounted with Vectashield mounting medium
plus 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories)
for viewing.
Superfusion culture. One-day-old male chicks were entrained
to a light/dark cycle for at least 7 d. Then the pineal glands were isolated and cultured individually in 10 mm wells of 48 well plates. The wells were sealed with silicon plugs that were equipped with inflow and outflow tubing. The culture chambers were kept at
38°C in 85% O2 and 5%
CO2, and culture medium (see above) was delivered
continuously to each chamber by a peristaltic pump at a flow rate of
350 µl/hr. The superfusion culture was started at a dark onset time
on the first day of culture. The chambers were kept in one cycle (the
first day) of 12 hr dark/light and then transferred to constant
darkness. Superfusate samples were collected every hour by a fraction collector.
Quantification of melatonin release by HPLC. Melatonin
levels in the superfusate samples were determined by NANOSPACE HPLC system (Shiseido, Tokyo, Japan) consisted of two 2001 inert pumps, a
2003 autosampler, a 2004 column oven, a 2009 degassing unit, a 2013 fluorometric detector, and a H-P valve. The HPLC system was equipped
with three columns; Capcell Pak C18 UG120 (1.5 × 150 mm;
Shiseido) for a main separation, Capcell Pak C18 UG120 (2.0 × 35 mm; Shiseido) as an intermediate column, and MF Cartridge column
(4.0 × 10 mm; Shiseido) for a deproteinization as described (Shirota et al., 1995). These columns were kept at 40°C. Superfusate samples were injected at a flow rate of 500 µl/min onto the first MF
Cartridge column equilibrated with Buffer P1 (100 mM
Na-phosphate and 5 mM sodium 1-octanesulfonate, pH 5.0).
Molecules with large molecular weights such as serum proteins pass
through the column, and small molecules such as melatonin are eluted
with retardation. This melatonin-containing fraction was introduced to
the intermediate Capcell Pak C18 column equilibrated with Buffer P1.
Molecules bound to this column were eluted with Buffer P2 (100 mM Na-phosphate and 5 mM sodium
1-octanesulfonate, pH 3.0) supplemented with 20% (v/v) acetonitrile at
a flow rate of 100 µl/min. The eluted molecules were separated by a
main Capcell Pak C18 column with Buffer P2 supplemented with 20% (v/v)
acetonitrile at a flow rate of 100 µl/min. The fluorescence intensity
of the final eluate was monitored continuously with excitation and
emission wavelengths set at 264 and 370 nm, respectively. Under the
chromatographic conditions, all metabolites produced from tryptophan in
the melatonin synthesis were separated from melatonin. Melatonin levels
were quantified by comparing the peak area of melatonin of samples with
those obtained from known concentrations of standards. For measurement of phase shifts, melatonin rhythms were smoothed by a three-point running average to minimize the effects of random measurement error on
phase determination. The times of half-rise and half-fall of the fourth
and fifth circadian peaks were measured as phase reference points for
each record. The phase shift was defined as the average of the
differences from control in the phase reference points (Cahill and
Besharse, 1993).
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RESULTS |
Light-induced dephosphorylation of MAPK in the chick
pineal gland
Chicks were entrained to a 12 hr light/dark cycle for 1 week and
were subsequently kept in constant darkness. The pineal glands were
isolated before and after exposure of chicks to light (for 15 min) at
circadian time (CT) 20, which induces a phase-advance (Takahashi et
al., 1989). Immunoblot analyses with anti-phosphotyrosine antibody
(Fig. 1a, left panel) demonstrated that the light
illumination caused a decrease in the level of tyrosine phosphorylation
of a 41 kDa pineal protein (pp41). This protein seemed to represent a
42 kDa serine-threonine kinase, MAPK [also called extracellular signal-regulated protein kinase (ERK)], because the pp41 migrated at
the upper edge of an anti-MAPK-immunoreactive band (Fig. 1a, right panel), which is known to undergo mobility shift (de
Vries-Smits et al., 1992) after dual phosphorylation of Thr-202 and
Tyr-204 (Anderson et al., 1990; Davis, 1993). To test this, MAPK was
immunoprecipitated from the chick pineal homogenate prepared at various
time points during the light exposure of animals at subjective night,
and the precipitates were immunoblotted with anti-phosphotyrosine antibody. As shown in Figure 1b, the level of tyrosine
phosphorylation of MAPK noticeably decreased within 20 min after the
onset of light at CT 18.0, whereas that of chicks kept in the darkness remained constant (Fig. 1b, CT 17.7 and 19.3). The MAPK
protein levels estimated by PanERK antibody (Fig. 1b) and
ERK2-specific antibody (data not shown) were almost constant during the
experiment, indicating that chick pineal ERK2 is dephosphorylated after
exposure of animals to light.
Circadian activation of chick pineal MAPK
In addition to the acute response to light, we observed an overt
rhythm in the phosphorylation state of pineal MAPK in constant darkness. As shown in Figure
2a (top
panel), chick pineal MAPK was tyrosine-phosphorylated
during subjective night with a peak at CT 16-20, and it becomes
dephosphorylated during subjective day without any significant change
in the protein level (bottom panel). Time-of-day
specific activation of MAPK (i.e., activation in subjective night)
caused by the dual phosphorylation was confirmed by an immune complex
kinase assay (Fig. 2b).
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Figure 2.
Circadian phosphorylation and activation of MAPK
in the pineal gland. Chicks entrained to the light/dark cycle for
7 d were transferred to constant darkness (day 8), and their
pineal glands were isolated every 4 hr on days 8 and 9. MAPK was
immunoprecipitated with anti-MAPK antibody from each pool of the pineal
homogenates. a, The immunoprecipitates were
immunoblotted with anti-phosphotyrosine antibody (top
panel) and then reprobed with anti-MAPK antibody
(bottom panel). Shaded and
solid horizontal bars at the bottom
indicate subjective day and night, respectively. b, The
immunoprecipitates at CT 8 and CT 16 were subjected to immune complex
kinase assay. The values are the means ± range of variation of
two independent measurements.
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To determine whether the activation rhythm of MAPK is associated with
the endogenous pacemaker in the chick pineal gland, the state of MAPK
phosphorylation was characterized in cultured pineal glands by using
anti-phospho-MAPK antibody, which specifically recognizes dually
phosphorylated (activated) MAPK. The rhythmicity of MAPK
phosphorylation was observed in culture under constant darkness (Fig.
3a, top panel) with a
peak at mid-to-late subjective night (CT 16-24), whereas MAPK protein
levels were nearly constant (bottom panel). The
rhythmicity persisted for at least 2 d in constant darkness (Fig.
3b), confirming that the molecular cycle reflects bona fide
circadian regulation. To localize the site of MAPK phosphorylation,
pineal sections (10 µm thickness) were prepared from the cultured
tissues at CT12 and 24 (representing inactivated and activated phases,
respectively; Fig. 3a), and they were subjected to
immunostaining by anti-phospho-MAPK antibody. No immunoreactivity was
observed in the pineal section prepared at CT 12, whereas at CT 24 strong immunoreactivities were found in the luminal layers of almost
all the follicles (Fig. 4). These follicular pinealocytes have pineal photoreceptor pinopsin (Okano et
al., 1994, 1997) and enzymes in melatonin biosynthesis, among which
N-acetyltransferase and
hydroxyindole-O-methyltransferase are known to colocalize
with the circadian pacemaker (Greve et al., 1993; Bernard et al., 1997;
Nakahara et al., 1997). Taken together, our observations strongly
suggest an intracellular linkage between circadian phosphorylation of
MAPK and the pacemaker.
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Figure 3.
Circadian rhythm in MAPK phosphorylation in
vitro. a, b, The pineal glands were cultured and
collected at the indicated circadian time in the dark, from CT 8 (a) or CT 24 (b) on the
second day of culture. Equal amounts of proteins (20 µg) were
immunoblotted with anti-phospho-MAPK antibody (top
panels) and then reprobed with anti-MAPK antibody
(bottom panels).
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Figure 4.
Localization of phosphorylated MAPK in the pineal
gland. Thin frozen sections were prepared from the cultured pineal
glands at CT 12 (left panels) or CT 24 (right
panels) on the second day of culture and immunostained with
anti-phospho-MAPK antibody. The nucleus was detected by staining with
DAPI. Scale bars, 50 µm.
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Involvement of MAPK in the circadian timing mechanism
Then we questioned the role of MAPK in a circadian rhythm
by examining the effect of pulse perturbation of pineal MAPK activity on the phase of the circadian clock. To this end, we used PD98059, a
reversible inhibitor of MAPK kinase (MEK) that is responsible for the
dual phosphorylation and activation of MAPK (Alessi et al., 1995).
Cultured chick pineal glands were treated with PD98059 for 12 hr from
Zeitgeber time (ZT) 12, and the level of MAPK phosphorylation was
investigated during and after the treatment in the darkness. Nighttime
phosphorylation of MAPK was inhibited by PD98059 in a dose-dependent
manner, and 80 µM PD98059 completely suppressed the phosphorylation
(Fig. 5a). When the drug was
removed, the phosphorylation level began to increase rapidly, peaking
at 4-8 hr after the removal, and then decreased. During this period, MAPK protein levels were nearly constant (data not shown). Such an
apparent shift of peak time in MAPK phosphorylation suggests a
drug-induced phase-shift of the circadian pacemaker. This idea was
tested by measuring circadian variations of two kinds of established outputs from the chick pineal pacemaker. One is a circadian variation in tryptophan hydroxylase (TRH) protein level in constant darkness (Florez and Takahashi, 1996). Eleven hour pretreatment (ZT 12-23) of
cultured pineal glands with 80 µM PD98059
induced a delay of the time-of-peak by 4-8 hr (Fig. 5b, bottom
panel) as compared to a control experiment (top
panel). In the other assessment of the effect of PD98059, a
circadian variation of the pineal melatonin production was measured by
using superfusion culture (Takahashi et al., 1980). To verify that
PD98059 specifically affects the circadian pacemaker, we examined
whether its effect is dependent on the circadian phase at which it is
presented. Then 12 hr treatment from CT 15 (Fig. 5c) induced
a clear phase-delay of 4.8 ± 1.3 hr (mean ± SE,
n = 3). In contrast, similar treatment during a frame
covering most of subjective daytime (CT 3-15) did not affect the phase
of the rhythm (Fig. 5d). This phase-dependent effect demonstrates that PD98059 is a genuine phase-shifting agent, and it is
unlikely that PD98059 serves to arrest cell activity. Similar circadian
variation in MAPK phosphorylation and the phase-shifting effect of
PD98059 were also observed in another clock-containing tissue, bullfrog
retina (Y. Harada, K. Sanada, and Y. Fukada, unpublished observations),
indicating that MAPK is an important component in the circadian timing
mechanism in vertebrates.
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Figure 5.
Effects of PD98059 on MAPK phosphorylation and the
circadian pacemaker. a, Cultured pineal glands were
treated with PD98059 (80 or 8 µM) in 0.1% DMSO for 12 hr
(ZT 12-24, gray area) on the first day of culture or
with 0.1% DMSO as a control. Pineal glands were collected at the
indicated time, and equal amounts of the pineal proteins (20 µg) were
immunoblotted with anti-phospho-MAPK antibody. Phosphorylated MAPK
levels were quantified by a densitometry. The means of two experiments
are shown in values relative to the band density of control (100%) at
ZT 18. b, Cultured pineal gland was treated with 80 µM PD98059 or DMSO for 11 hr from ZT 12 as in
a. Pineal glands were collected every 4 hr on the second
and third day of culture, and each pool of the homogenates (20 µg
proteins) was immunoblotted with anti-TRH antibody. TRH levels were
quantitated by a densitometry and shown in values relative to the peak
value set to 100%. The data are representative results of replicate
experiments with similar results. c, d, Pineal glands
were cultured individually in superfusion culture chambers and were
treated with 80 µM PD98059 (closed circles) in 0.1%
DMSO or with 0.1% DMSO (open circles) for 12 hr from CT
15 (c) or CT 3 (d) in
constant darkness. Melatonin levels in the superfusate samples were
determined by HPLC. Melatonin levels for each pineal gland were
shown in values relative to the average melatonin release on the second
day of culture. The data are representative results of three
independent pairs of experiments. The treatment with PD98059 from CT 15 (c) induced a phase-delay of 4.8 ± 1.3 hr
(mean ± SE, n = 3), whereas that from CT 3 (d) induced no significant phase-shift
(n = 3).
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DISCUSSION |
Our results provide the first evidence that MAPK contributes to
circadian time keeping in the chick pineal glands. The phosphorylation rhythm of MAPK seems to reflect an overall oscillation of the MAPK
cascade, because we observed a clear circadian oscillation in kinase
activities of MEK and Raf in the chick pineal gland (Y. Hayashi, K. Sanada, and Y. Fukada, unpublished observations). It is possible that
some of upstream regulators of the MAPK cascade are controlled at the
transcriptional level by an autoregulatory feedback loop (Takahashi,
1995; Dunlap, 1998; Reppert, 1998), and that MAPK mediates feedback of
the output back to input, forming a secondary loop. These
interconnected loops could determine the circadian properties such as
period length (Dunlap, 1998, 1999). Alternatively, MAPK cascade may be
activated by intercellular communication and mediate coupling of
pacemakers among clock cells. In the circadian feedback loop, some of
the essential clock components exhibit a daily/circadian rhythm in the
level of phosphorylation (Zeng et al., 1996; Lee et al., 1998). MAPK
might be one of the upstream kinases responsible for phosphorylating
and regulating the biochemical activities (and stabilities) of the
clock proteins or other components such as cAMP response
element-binding protein, which has been implicated in circadian
regulation of period gene expression (Belvin et al.,
1999).
Recently it was reported that MAPK phosphorylation exhibited a
circadian variation in the mouse SCN (Obrietan et al., 1998), although
its involvement in the pacemaker machinery was not evaluated. Noticeably, the circadian rhythmicity of MAPK phosphorylation in the
mouse SCN had a peak during subjective day (Obrietan et al., 1998),
whereas those in the chick pineal gland (Fig. 2, 3) and frog retina (Y. Harada, K. Sanada, and Y. Fukada, unpublished observations) did during
subjective night. A more marked contrast is the acute effect of light;
that is, MAPK in the chick pineal gland (Fig. 1) is rapidly
dephosphorylated after nighttime light illumination, which induces
inversely phosphorylation of MAPK in the mouse SCN. Despite such a
contrast, the MAPK cascade may be involved in a common oscillator
function in vertebrate clock systems. Signal transduction pathways
upstream and downstream of the MAPK cascade and regulation of MAPK
phosphatase are to be investigated for better understanding of the
relationship between the MAPK cascade and the circadian oscillation
mechanism in vertebrates.
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FOOTNOTES |
Received July 19, 1999; revised Nov. 5, 1999; accepted Nov. 11, 1999.
This work was supported in part by Grants-in-Aid from the Japanese
Ministry of Education, Science, Culture, and Sports.
Correspondence should be addressed to Dr. Yoshitaka Fukada, Department
of Biophysics and Biochemistry, Graduate School of Science, The
University of Tokyo, Hongo 7-3-1, Bunkyo-Ku, Tokyo 113-0033, Japan.
E-mail: sfukada{at}mail.ecc.u-tokyo.ac.jp.
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ABSTRACT
INTRODUCTION
