CKI␧/␦-dependent phosphorylation is atemperature-insensitive, period-determining
process in the mammalian circadian clock
Yasushi Isojimaa,b,1, Masato Nakajimac,1, Hideki Ukaic,1, Hiroshi Fujishimac, Rikuhiro G. Yamadac, Koh-hei Masumotoc,
Reiko Kiuchia,b, Mayumi Ishidac, Maki Ukai-Tadenumac, Yoichi Minamic, Ryotaku Kitoc, Kazuki Nakaod,
Wataru Kishimotoc, Seung-Hee Yooe,f,g, Kazuhiro Shimomuraf,h, Toshifumi Takaoi, Atsuko Takanoj, Toshio Kojimaa,
Katsuya Nagaij, Yoshiyuki Sakakia, Joseph S. Takahashie,f,g,h,2, and Hiroki R. Uedac,k,l,2
aComparative Systems Biology Team, Genomic Science Center and bDevelopmental Systems Modeling Team, Advanced Computational Sciences Department,

RIKEN, 1-7-22, Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; cLaboratory for Systems Biology, kFunctional Genomics Unit, and dAnimal Resource Unit,
RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan; eHoward Hughes Medical Institute, fDepartmentof Neurobiology and Physiology, and hCenter for Functional Genomics, Northwestern University, 2205 Tech Drive, Evanston, IL 60208; gDepartment of
Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390-9111; iLaboratory of Protein Profiling and Functional Proteomics,
and jLaboratory of Proteins Involved in Homeostatic Integration, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871,
Japan; and lDepartment of Bioscience, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan

A striking feature of the circadian clock is its flexible yet robust
response to various environmental conditions. To analyze thebiochemical processes underlying this flexible-yet-robust characteristic, we examined the effects of 1,260 pharmacologically active
compounds in mouse and human clock cell lines. Compounds thatmarkedly (>10 s.d.) lengthened the period in both cell lines, also
lengthened it in central clock tissues and peripheral clock cells.
Most compounds inhibited casein kinase I␧ (CKI␧) or CKI␦ phosphorylation of the PER2 protein. Manipulation of CKI␧/␦dependent phosphorylation by these compounds lengthened theperiod of the mammalian clock from circadian (24 h) to circabidian(48 h), revealing its high sensitivity to chemical perturbation. The
degradation rate of PER2, which is regulated by CKI␧/␦-dependent
phosphorylation, was temperature-insensitive in living clock cells,
yet sensitive to chemical perturbations. This temperature-insensitivity was preserved in the CKI␧/␦-dependent phosphorylation ofa synthetic peptide in vitro. Thus, CKI␧/␦-dependent phosphorylation is likely a temperature-insensitive period-determining process in the mammalian circadian clock.
chemical biological approach 兩 temperature compensationT

he circadian clock is a molecular mechanism underlying
endogenous, self-sustained oscillations with a period of ⬇24h, manifested in diverse physiological and metabolic processes(1–3). The most striking feature of circadian clock is its flexible
yet robust response to various environmental conditions. For
example, circadian periodicity varies with light intensity (4–6)
while remaining robust over a wide range of temperatures(‘‘temperature compensation’’) (1, 3, 7–9). This flexible-yetrobust characteristic is evolutionarily conserved in organisms
ranging from photosynthetic bacteria to warm-blooded mammals (3, 10–12), and has interested researchers from a broad
range of disciplines. However, despite many genetic and molecular studies (13–22), the detailed biochemical mechanism underlying this characteristic remains poorly elucidated (3).
The simplest explanation for this flexible-yet-robust property
is that the key period-determining reactions are insensitive totemperature but responsive to other environmental conditions.
Indeed, Pittendrigh proposed the existence of a temperatureinsensitive component in the clock system in 1954 (7), and in
1968, he and his colleagues demonstrated that both the wave
form and the period of circadian oscillations are invariant withtemperature (23). However, the idea of a temperatureinsensitive biochemical reaction is counterintuitive, as elementary chemical processes are highly temperature-sensitive. One
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0908733106

exception is the cyanobacterial clock, in which temperatureinsensitive enzymatic reactions are observed (24, 25). However,the cyanobacterial clock is quite distinct from other clock
systems, and this biochemical mechanism has not been demonstrated in other clocks.
Recently, a chemical-biological approach was proposed tohelp elucidate the basic processes underlying circadian clocks(26), and high-throughput screening of a large chemical compound library was performed (27). In this report, to analyze
systematically the fundamental processes involved in determining the period length of mammalian clocks, we tested 1,260
pharmacologically active compounds for their effect on period
length in mouse and human clock cell lines, and found 10
compounds that most markedly lengthened the period of bothclock cell lines affected both the central and peripheral circadianclocks. Most compounds inhibited CKI␧ or CKI␦ activity, suggesting that CKI␧/␦-dependent phosphorylation is an important
period-determining process in the mammalian circadian clock.
Surprisingly, the degradation rate of endogenous PER2, whichis regulated by CKI␧-dependent phosphorylation (28) and probably by CKI␦-dependent phosphorylation, was temperatureinsensitive in the living clock cells, and the temperatureinsensitivity was preserved even for the in vitro CKI␧/␦dependent phosphorylation of a synthetic peptide derived from
PER2. These results suggest that this period-determining process is flexible in response to chemical perturbation yet robust in
the face of temperature perturbations. Based on these findings,
we propose that CKI␧/␦-dependent phosphorylation is a temperature-insensitive period-determining process in the mammalian circadian clock.
Results
Ten Compounds Markedly Lengthened Period Length. We examined
1,260 pharmacologically active chemical compounds from the

Author contributions: Y.I., M.N., H.U., T.T., K. Nagai, and H.R.U. designed research; Y.I.,
M.N., H.U., H.F., K.-h.M., R. Kiuchi, M.I., M.U.-T., Y.M., R. Kito, K. Nakao, W.K., S.-H.Y., K.S.,
A.T., T.K., Y.S., and J.S.T. performed research; W.K. contributed new analytic tools; R.G.Y.
analyzed data; and Y.I., M.N., H.U., and H.R.U. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1Y.I., M.N., and H.U. contributed equally to this work.
2To

whom correspondence may be addressed. E-mail: uedah-tky@umin.ac.jp or
j-takahashi@northwestern.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/
0908733106/DCSupplemental.

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contributed by Joseph S. Takahashi, August 4, 2009 (sent for review June 14, 2009)

LOPAC1280 library for their effect on circadian period length inmammalian clock cell lines, NIH 3T3-mPer2-Luc and U2OShPer2-Luc (Table S1 and Table S2 and SI Text). We identified 10,
all with period-lengthening effects, that substantially altered theperiod in both cell lines (⬎ ⫹10 s.d. or ⬍ ⫺10 s.d., ⬇2.0–3.0 h)(Fig. S1 A and D and Tables S3 and S4). These compounds,
labeled ‘‘potent,’’ also lengthened the period of primary culturesof mouse embryonic fibroblasts (MEFs, as peripheral clock cells)(Fig. S1 B and E) and slice cultures of suprachiasmatic nucleus(SCN; as central clock tissue) (Fig. S1 C and E). These results
supported the idea that the potent compounds alter canonicalclock processes.
Potent Compounds Inhibit CKI␧/␦ Enzymatic Activity. Most of the
proposed targets of the 10 potent compounds on the list in Table
S3 have not been identified as components of the mammalian
circadian clock, although one of the 10 is an inhibitor of CK2,
recently found as a component of the mammalian circadian clock(29, 30). The potent protein kinase inhibitors SB202190,
SP600125, and roscovitine were previously reported to inhibit
CKI in addition to their primary targets (31, 32). Since evenspecific protein kinase inhibitors also usually affect other kinases(31, 33), we hypothesized that the seven protein kinase inhibitorsthat we identified suppress CKI activity in addition to theirprimary targets.
To examine this hypothesis, we performed the siRNA knockdown of CKI genes, as well as the genes related to the 10 potent
compounds (Fig. 1 A–C and Fig. S2 B–G). The knockdown of
CKI␧ or CKI␦ exhibited the greatest period-lengthening effects(Fig. 1B), more than 28 h in CT, whereas knockdown of othergenes showed no or only a slight effect (Fig. S2 B–G). Moreover,the combinatorial knockdown of CKI␧ and CKI␦ additively
lengthened the period of circadian oscillations to over 30 h in CT(Fig. 1C), supporting the idea that CKI␧/␦ play one of the mostimportant roles in the period-determination processes of mammalian circadian clocks. These results suggest that at least someof our compounds were not acting via their primary target, but
through inhibition of CKI␧/␦.
The inhibitory effect of the potent compounds on CKI␧/␦
activity was confirmed by the in vitro CKI␧ or CKI␦ kinase assay.
Importantly, nine of the 10 compounds [i.e., not 17 ␣ hydroxyprogesterone (17-OHP)] strongly inhibited the catalytic
domain of wild-type CKI␧, lacking the C-terminal regulatory
domain (⌬CKI␧) as effectively as or better than IC261, a specificinhibitor of CKI (Fig. 1D). SP600125 and TG003, which dramatically extended the circadian period and potently inhibited⌬CKI␧ activity, strongly inhibited both activity of the ⌬CKI␧ andcatalytic domain of CKI␦ (⌬CKI␦), with lower IC50 (⬍ 0.55 ␮M)than the IC50 of IC261 (about 4 ␮M), whereas 17-OHP did notinhibit both of ⌬CKI␧ and ⌬CKI␦ (Fig. 1E). Since double
knockdown of the CKI␧ and CKI␦ genes using siRNA had
additive effects on period lengthening (Fig. 1C), and thesechemical compounds, except 17-OHP, inhibited CKI␧ and CKI␦
similarly, they probably acted on both CKIs to affect the period
length of circadian oscillations.
We also examined the effect of potent compounds on thestability of the mPER2 protein, which was regulated by CKI␧(28) (and probably by CKI␦) in non-clock cell, 293T cells. Fig. S3
A and B show that the two putative CKI inhibitors significantly
enhanced the stability and slowed the degradation rate of
LUC::mPER2 (P ⬍ 0.01, one-way ANOVA), whereas 17-OHP
did not significantly affect LUC::mPER2 stability (P ⫽ 0.18,
one-way ANOVA). These results were supported by immunoblot
experiments (Fig. S4). This degradation rate of overexpressed
LUC::mPER2 was not affected without the co-overexpression of
CKI␧(tau) (Fig. S3C; P ⫽ 0.193 for SP600125 and P ⫽ 0.728 for
TG003; two-way ANOVA), presumably because relative expression levels of LUC::mPER2 in 293T cells compared with CKI␧/␦
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Fig. 1. Effects of knockdowns of CSNK1s on the period length and effect ofpotent compounds on the CKI␧/␦ activity in vitro. (A–C) Graphs indicate
relationship between gene knockdown effects and period length in U2OShPer2-Luc cells. The x-axis indicates the expression level of genes relative to thesamples transfected with control siRNA. The y-axis indicates the period length,
described in circadian time (CT), with the control samples assigned as 24 h.
Each symbol represents the mean ⫾ SEM of independent experiments (n ⱖ 3).
(A) The effect of CRY2 knockdown as a positive control. (B) Gene knockdownsof CKI␧/␦. CSNK1E, CKI␧; CSNK1D, CKI␦. (C) Effect of the double knockdown of
CSNK1E and CSNK1D. The gene expression level represents the total amountof CKI␦ and CKI␧. (D) ⌬CKI␧ phosphorylation activity for a synthetic mPER2
peptide in the presence of chemical compounds [25, 50, or 100 ␮M for the 10compounds, 100 ␮M for IC261] was measured using a modified IMAP assay
with 100 ␮M ATP. The average results for each condition are shown as therelative activity compared to the control condition (DMSO). Error bars denote
1 s.d. (E) Dose-dependent effects of SP600125 and TG003 on ⌬CKI␧/␦ phosphorylation activity. ⌬CKI␧/␦ phosphorylation activity was measured in thepresence of compounds at the indicated concentrations. Each symbol indicates the activity relative to the control condition in two independent experiments. The lines are approximate functions using the equation: y ⫽ 100e-ax.
The IC50s for CKI␧ calculated from the equations were 4.0, 0.55, and 0.22 ␮M
for IC261, TG003, and SP600125, respectively, and for CKI␦ were 4.1, 0.40, and0.13 ␮M.

were much higher that in MEFs. We used CKI␧(tau) for theseassays because the degradation rate with co-overexpression of
CKI␧(tau) was faster than that of CKI␧(wt), which enable us tomeasure the PER2 degradation rate precisely. We next confirmed that the stability of LUC-fused mPER1 protein(LUC::mPER1), the closest relative of mPER2, was also decreased with co-expression of CKI␧(tau) as well as
LUC::mPER2,
whereasLUC-fused
mBMA L1(LUC::mBMAL1) and native LUC were not (Fig. S5). These
results suggest that the inhibitory effect of the potent compounds
on CKI␧/␦ activity is the primary mechanism by which theylengthened the period of clock cells. In contrast, 17-OHP
apparently functions via a different mechanism.
Flexibility and Robustness of a Period Determination Process. To

explore how important this process is for determining the period
Isojima et al.

length of the mammalian circadian clock, we examined theeffects of these two compounds on the circadian period length
at higher concentrations (50–500 ␮M). Interestingly, the higher
concentrations approximately doubled the period length (to53.98 and 52.50 h at 100 ␮M for TG003, and 48.63 and 49.74 h
at 500 ␮M for SP600125) relative to the control U2OS-hPer2Luc cells (26.89 and 27.02 h) (Fig. 2A). Therefore, these results
suggest that a period-determining process mediated by CKI␧/␦Isojima et al.

CKI␧/␦ Activity Is Insensitive to Temperature. To examine thebiochemical foundation underlying the observed temperatureinsensitivity, we analyzed the phosphorylation activity of CKI␧and CKI␦ in vitro. To facilitate this analysis, we designed asynthetic peptide substrate derived from the putative ␤TrCPbinding region of mouse PER2 (referred to here as ␤TrCPpeptide) (Fig. S6A), because this region is important for phosphorylation-dependent degradation of PER2 in the mammalianclock system (34). We first used just the catalytic domain of
wild-type CKI␧, lacking the C-terminal regulatory domain[⌬CKI␧(wt)] to prevent potential confusion that could result
from the autophosphorylation of this regulatory domain and thesubsequent repression of CKI␧ kinase activity. This use of thecatalytic domain was also justified by evidence that CKI␧ is kept
in a dephosphorylated, active state in vivo (35).
Under this experimental setup, we successfully recapitulated,
at least partially, the enhanced phosphorylation seen with a⌬CKI␧ tau mutation [⌬CKI␧(tau)] (36) in comparison with
⌬CKI␧(wt) at 35 °C (Fig. 3A, red). Consistent with our otherfindings, ⌬CKI␧(wt) and ⌬CKI␧(tau) phosphorylated the peptide substrate at similar rates whether at 25 °C (Fig. 3A, blue) or
35 °C, indicating a strong temperature-insensitivity (Q10 ⫽ 1.0)
[Fig. 3D, ⌬CKI␧(wt) and ⌬CKI␧(tau)]. Similar temperatureinsensitivity were also observed by using catalytic domain of
wild-type CKI␦ lacking the C-terminal regulatory domain[⌬CKI␦(wt)] (Q10 ⫽ 1.2) [Fig. 3D, ⌬CKI␦].
The temperature-insensitivity of the CKI␧ reaction depended
substantially on the phosphorylation state of CKI␧ itself, because
the Q10 of the autophosphorylated full-length construct (preincubated with ATP) was greater than for the isolated catalytic
domain (Fig. 3 B and D, CKI␧, preincubated). We also confirmed an earlier report that preincubating full-length CKI␧ with
ATP repressed its enzymatic activity (⬇15-fold and 8-fold at 25 °and 35 °C, respectively) (37); this was probably due to the
autophosphorylation of the C-terminal regulatory domain (Fig.
S6 B and C). The effects of autophosphorylation on enzymatic
activity were also recapitulated by a mutant in which the eight
autophosphorylated serine residues in the C-terminal regulatory
domain (37) were replaced with glutamate [Fig. 3D, CKI␧(D8)].
Using this autophosphorylation mutant, we observed both aslightly enhanced temperature sensitivity [Q10 ⫽ 1.3, an increase
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Fig. 2. Flexibility and robustness of a period-determination process of the
mammalian circadian clock. (A) Dose-dependent effects of SP600125 andTG003 on period length in U2OS-hPer2-Luc cells. The period length is indicatedboth in real-time (right axis) and in circadian time (left axis). For circadian time,
the average period length in two independent control experiments was
assigned as 24.0 h. The two lines in each graph correspond to two independent
experiments. Each value represents the mean ⫾ SEM. At the concentrations
without data points, the cells behaved arrhythmically. (B and C) Dosedependent effect of SP600125 on the period length and degradation rate ofmPER2::LUC in mPer2Luc MEFs. A pair of plates with cultured mPer2Luc MEFs, towhich 0 to 10 ␮M SP600125 was applied, were prepared. One was used tomeasure mPER2::LUC decay and the other to determine the period. (B) Decay
of mPer2::LUC bioluminescence in mPer2Luc MEFs. The degradation ofmPer2::LUC protein was monitored after the administration of CHX to MEFs.
The time-course data of each sample were normalized to approximate functions in which time point 0 was 100%. Each value represents the mean ⫾ SEM.
of the normalized data. The lines represent approximated curves in which y ⫽100 at time ⫽ 0 and y ⫽ 50 at the averaged half-life time. The colors in orderfrom gray to blue to red represent the concentration of SP600125 with 0.25%
DMSO (n ⫽ 6). (C) Correlation between the period length and degradation
rate of mPER::LUC in mPer2Luc MEFs with the administration of SP600125. Eachvalue represents the mean ⫾ SEM (n ⫽ 6). (D) Temperature dependency of
decay of the mPER2::LUC bioluminescence in mPer2Luc MEFs. The degradationof mPER2::LUC protein was monitored after the addition of CHX to MEFs. The
time-course data of each sample were normalized to an approximate functionin which time point 0 was 100%. Each value represents the mean ⫾ SEM of thenormalized data. The lines represent an approximated curve in which y ⫽ 100
at time ⫽ 0 and y ⫽ 50 at the averaged half-life time. The blue dots and line
indicate the data at 27 °C; green, 32 °C; and magenta, 37 °C (n ⫽ 23). (E)Temperature compensation in the half-lives of the mPer2::LUC protein. The
graph indicates the mean ⫾ SEM. The gray broken line indicates the approximated line described by the equation: y ⫽ 53.69 ⫹ 0.333x, and the Q10 value
between 27 and 37 °C calculated from the equation is 0.950. (F) Temperaturecompensation in the period length of mPer2Luc MEFs. The graph indicates themean ⫾ SEM. The gray broken line indicates the approximated line describedby the equation: y ⫽ 19.02 ⫹ 0.097x, and the Q10 value between 27 and 37 °C
calculated from the equation is 0.957.

dependent phosphorylation is remarkably flexible to chemical
perturbation, since a single compound lengthened the period
from circadian (⬇24 h) to almost circabidian (⬇48 h).
To further confirm this flexibility, we next investigated thesensitivity of this process to chemical perturbation in living clock
cells by using mPer2Luc MEFs. We observed that the period
length of the circadian oscillation in MEFs correlated well withthe mPER2::LUC stability under the administration of a potent
compound (Fig. 2 B and C). Given this strong correlation, we
concluded that CKI␧/␦ activity on the PER2 protein is one of themost important period-determining processes in the mammalian
circadian oscillator.
If important period-determination processes were highly sensitive to temperature, it would be very difficult to maintain thetemperature compensation over the entire circadian period.
Thus, we next investigated the temperature dependency of thisprocess in living clock cells by using mPer2Luc MEFs. We foundthat the degradation rate of mPER2::LUC and the period length
were completely temperature-insensitive in the MEFs (Fig. 2
D–F). The observed temperature-insensitivity seemed to reflect
a molecular property of the endogenous mPER2 protein, because the degradation rate of LUC is sensitive to temperature in
mammalian cells (Fig. 4 A and B). These results imply that theperiod-determination process, which was sensitive to chemical
perturbation, was remarkably robust against physical perturbation such as temperature differences.

Fig. 3. Temperature insensitivity of the CKI␧/␦ phosphorylation activity. (A) Temperature dependency of the ⌬CKI␧(wt) (circles) and ⌬CKI␧ (tau) (squares)
phosphorylation activity for the ␤TrCP-peptide substrate. Assays were performed at 25 °C (blue) and 35 °C (red). (B) Phosphorylation activity of full-length
CKI␧(wt) autophosphorylated by preincubation with ATP. Assays were performed at 25 °C (blue) and 35 °C (red). (C) Temperature dependency of the full-length
CKI␧ phosphorylation activity for the ␤TrCP-peptide substrate. Phosphorylation activity of full-length CKI␧(wt) (CKI␧, solid line), and CKI␧(wt) dephosphorylatedwith ␭ protein phosphatase (CKI␧, dephosphorylated) (broken line). Assays were performed at 25 °C (blue) and 35 °C (red). (D) Summary for temperaturedependency of CKI␧/␦ enzymatic activities.

of 0.3 compared with ⌬CKI␧(wt)] and a repression of enzymatic
activity (⬇6-fold and ⬇5-fold at 25 ° and 35 °C, respectively). We
confirmed these effects on the time-course of phosphorylation
by full-length CKI␧ that was not preincubated with ATP: As the
incubation time increased (and probably as autophosphorylation
increased), the enzymatic activity slowed (slopes of the curves
decreased), and the temperature sensitivity (i.e., the differencein slope between 25 ° and 35 °C) increased (Fig. 3C, CKI␧). We
further confirmed these effects on the time course of phosphorylation using dephosphorylated full-length CKI␧ without preincubation (Fig. S6 B and C) and found that the enzymatic activity
slowed substantially and the temperature sensitivity increased
between the first hour and the next 3 h (Fig. 3C, CKI␧,dephosphorylated). The effect of autophosphorylation on thetemperature dependency was more prominent with CKI␧ thanwith the closely related CKI␦; the repression of enzymatic
activity was, however, also observed for CKI␦ (Fig. 3D, ⌬CKI␦
and CKI␦, preincubation).
PER2 phosphorylation is a complicated process involving
multiple phosphorylation sites (38). To explore the temperaturedependency of CKI␧ with another substrate, we designed asynthetic peptide substrate derived from the region near thefunctional amino acids in PER2, which is responsible for familial
advanced sleep phase syndrome (FASPS) (referred to here as
FASPS-peptide) (Fig. S6A). We chose this site because a serine
at position 662 of human PER2 (amino acid 659 of mPER2) was
identified as a mutation for FASPS (39). We found that thephosphorylation of FASPS-peptide by ⌬CKI␧(wt) was also
temperature-insensitive, with a Q10 ⫽ 1.2, whereas the phosphorylation by ⌬CKI␧(tau) was more temperature-sensitive(Q10 ⫽ 1.6) (Fig. S6D). These results with the FASPS- and␤TrCP-peptides suggest that CKI-dependent phosphorylation
can be temperature-insensitive, depending on both the substrate
and the state of the enzyme.
Since CKI␧/␦ is involved in a variety of other biologicalprocesses, we next investigated the temperature dependency of
its activity for two clock-unrelated substrates: casein, a conven4 of 6 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0908733106

tional protein substrate, and CK1tide, a commercially available
peptide substrate for CKI␧/␦, which was a peptide substrate
phosphorylated at two residues N-terminal of CKI␧/␦ phosphorylation site. Enhanced temperature sensitivity was observed forthese substrates (Q10 ⫽ 1.6 for casein and 1.4 for CK1tide) (Fig.
3D), although it was still less than expected for canonicalenzymatic reactions (Q10 ⫽ 2–3). As with ␤TrCP-peptide,enhanced temperature sensitivity due to autophosphorylation
was also observed for these substrates (Q10 ⫽ 2.4 for casein, an
increase of 0.8; and Q10 ⫽ 2.0 for CK1tide, an increase of 0.6)
(Fig. 3D), confirming that the temperature insensitivity depends
substantially on the substrate as well as on the state of the
enzyme (see also SI Text).
Although we demonstrated that the enzymatic activity of
CKI␧ was temperature-insensitive for two synthetic peptide
substrates (␤TrCP-peptide and FASPS-peptide) derived from
mPER2 as described above, it was still unclear if this temperature
insensitivity would hold for the full-length mPER2 protein,which may have multiple phosphorylation sites outside theputative ␤TrCP-binding region and FASPS site. We therefore
examined the degradation rate of mPER2::LUC, which wasoverexpressed in mouse clock cells (NIH 3T3) and compared it
to that of LUC. As expected, the degradation rate of LUC was
highly sensitive to temperature between 27 ° and 32 °C (Q5 ⫽2.08 and 2.10, extrapolated Q10 ⫽ 4.33 and 4.41, without or with
⌬CKI␧, respectively; Fig. 4 A, B, and E). The Q5 represent therelative change of a physical property as a consequence ofincreasing the temperature by 5 °C. On the other hand, thedegradation rate of mPER2::LUC was less sensitive to temperature (Q5 ⫽ 1.51, extrapolated Q10 ⫽ 2.28; Fig. 4 C and E).
Importantly, enhancing the enzymatic reaction between mPER2
and CKI␧ by additionally expressing the catalytic domain of
CKI␧ (⌬CKI␧) in NIH 3T3 cells recapitulated the temperature
insensitivity of the mPER2::LUC degradation rate (Q5 ⫽ 1.22,
extrapolated Q10 ⫽ 1.49; Fig. 4 D and E). Based on these results
together with our finding in mPer2Luc MEFs (Fig. 2 D–F), we
concluded that CKI␧/␦-dependent phosphorylation of the
Isojima et al.

which is regulated by CKI␧- or CKI␦-dependent phosphorylation, was temperature-insensitive in the living clock cells (Fig. 2
D and F). We also found that temperature insensitivity waspreserved even in the CKI␧/␦-dependent phosphorylation of asynthetic peptide derived from mPER2 in vitro (Fig. 3). Moreover, the expression of exogenous full-length mPER2 protein
and the catalytic domain of CKI␧ in NIH 3T3 cells recapitulated
the temperature-insensitive reaction (Fig. 4). Based on theseexperimental data, we propose that CKI␧/␦-dependent phosphor ylation is likely a temperature-insensitive perioddetermining process in the mammalian circadian clock. In
particular, we note that this enzymatic reaction is a periodaccelerating reaction in mammalian circadian clocks, because
enhancing this reaction by genetic mutation leads to shorteningof the circadian period (36, 40), and because pharmacologicalinhibition of this reaction led to lengthening of the circadianperiod of human clock cells from a circadian (⬇24 h) to almost
circabidian (⬇48 h) period.

Fig. 4. Expression of full-length mPER2 and a catalytic domain of CKI␧ in NIH
3T3 cells recapitulates the temperature-insensitive reaction. (A–D) Temperature dependency of decay of LUC (A and B) or mPer2::LUC (C and D) bioluminescence in NIH 3T3 cells. NIH 3T3 cells, transfected with reporter vector(pMU2-Luc or pMU2-mPer2::Luc) and an expression vector for ⌬CKI␧ (B and D)or empty vector (A and C), were used. The degradation of LUC or mPer2::LUC
was monitored after the administration of CHX to cells. The time-course dataof each sample were normalized to approximate functions in which time point
0 was 100%. Each value represents the mean ⫾ SEM of the normalized data.
The lines represent an approximated curve in which y ⫽ 100 at time ⫽ 0 andy ⫽ 50 at the averaged half-life time. Blue dots and line indicate the data at
27 °C; and green, 32 °C (n ⫽ 6 –11). (E) Q5 values (the ratio between 27 and
32 °C) of the degradation rate of LUC or mPER2::LUC with or without coexpression of ⌬CKI␧. The half-lives of LUC or mPER2::LUC in each sample were
calculated as described in the SI Materials and Methods.

mPER2 protein, which can determine the period length ofcircadian oscillations in clock cells, is likely temperatureinsensitive.
Discussion
A Period-Determining and Temperature-Insensitive Process. We discovered a highly flexible period-lengthening response in the
mammalian clock: A human clock cell’s circadian period could
be stretched into a circabidian period (Fig. 2 A and E) throughchemical manipulation, probably of the CKI activity, by
SP600125 and TG003. We found that the knock-down of
CSNK1E and CSNK1D had strong period-lengthening effects,
but that of other kinases had much weaker effects, if any (Fig.
1 A–C and Fig. S2 B–G). Although we cannot completely exclude
the possibility that our identified compounds inhibit otherenzymes involved in mammalian circadian oscillations, we reason, as a first-order approximation, that these compounds
lengthen the mammalian circadian oscillation primarily by inhibiting the CKI␧ and CKI␦ enzymatic activity (see also SI Text).
We also found that this chemically f lexible perioddetermination process is highly robust against temperature
differences, because the degradation of endogenous mPER2,
Isojima et al.

observed period-accelerating and temperature-insensitive reaction is consistent with the previously proposed general theory forthe temperature compensation of circadian clocks (41). In 1957,
Sweeney and Hastings proposed that the temperature insensitivity of the primary period-determining reaction might beachieved through a balance between multiple, counteracting
chemical reactions (8). This balance idea, which was mathematically formulated and generalized by Ruoff in 1992 into thebalance theory (41), describes a system in which there is abalance between period-accelerating and period-decelerating
reactions that yields the temperature-compensated circadianoscillation. In detail, if the period-accelerating and perioddecelerating reactions were equally sensitive to temperature, thecircadian oscillation would get faster with an increase in temperature. To reconcile this issue, the balance theory proposesthat the period-accelerating reaction(s) are more temperatureinsensitive than the period-decelerating reaction(s), and, hence,these two sets of enzymatic reactions are balanced to maintaina constant circadian oscillation. In an extreme and ideal case, thebalance theory predicts that the period-accelerating reaction
would be temperature-insensitive (41), which was suggested bythis study.
A Period-Accelerating and Temperature-Insensitive Reaction Is an
Evolutionarily Conserved Design Principle from Cyanobacteria to
Mammals. This observed temperature insensitivity of an enzy-matic reaction is the second example of such a characteristic ina circadian system. A similar temperature insensitivity wasrecently observed in the evolutionarily distinct cyanobacterial
clock system (Q10 ⫽ ⬇1.3 for autophosphorylation of the
cyanobacteria clock protein KaiC) (24). This remarkable similarity between two very different systems implies that temperature-insensitive enzymatic reactions might represent an evolutionarily conserved design principle. We do not insist here that
all individual period-determining reactions in the mammalian
circadian clock are temperature-insensitive, as is observed in the
cyanobacteria clock. Rather, we propose that there are sometemperature-insensitive period-determining enzymatic reactions in the mammalian circadian clock, which is consistent withthe balance theory. Since biochemical reactions have been
believed to be highly sensitive to temperature differences for along time, our results on the existence of temperature-insensitive
reaction in vitro and in cellulo, with possible implications fortemperature compensation of circadian clocks, suggest the surprising capability of CKI␧/␦-dependent phosphorylation. Therefore, remaining challenges are to obtain the atomic resolution
model of this temperature-insensitive reaction, as well as to
PNAS Early Edition 兩 5 of 6

CELL BIOLOGYA Period-Accelerating and Temperature-Insensitive Reaction Is Consistent with a General Theory of Temperature Compensation. The

demonstrate the physiological relevance of this temperatureinsensitive reaction in circadian temperature compensation. Ourin vitro temperature-insensitive reaction system, consisting of ashort peptide, ␤TrCP-peptide, and a monomeric enzyme, CKI␧or CKI␦, would be an ideal experimental platform, which will
provide atomic and molecular information required to test thephysiological relevance of this temperature-insensitive reaction
in circadian temperature compensation.
Materials and Methods
Analysis of the Effects of Compounds on Period Length in Cultured Cells. NIH
3T3-mPer2-Luc cells and U2OS-hPer2-Luc cells were cultured on 24-well(PerkinElmer), 96-well, or 384-well (Falcon) culture plates in medium supplemented with 200 ␮M luciferin (Promega) and a chemical compound withstimulation with 10 nM forskolin (Nacalai Tesque). The time-course bioluminescence data were analyzed as reported previously (42).

assay, the full-length CKI␧ and CKI␦, the catalytic domain of CKI␧, CKI␦, and thetau-mutant of CKI␧ [⌬CKI␧, ⌬CKI␦, ⌬CKI␧ (tau)], and a mutant in which theeight autophosphorylated serine residues in the C-terminal regulatory domain were replaced with glutamate [CKI␧(D8)] purified from bacterial lysate
were used as the enzymes in this assay.
Analysis of Temperature Sensitivity of mPER2 or LUC Degradation in NIH 3T3
Cells. NIH 3T3 cells were transfected with reporter vector (pMU2-mPer2::Luc or
pMU2-Luc) and pMU2-⌬CKI␧(wt) or empty pMU2 using FuGene6. The transfected cells were harvested by trypsin and plated on 24-well culture plates 24 hafter transfection, and the degradation of mPER2::LUC or LUC was measured
as described above for mPer2Luc MEFs.

Measurement of CKI␧ and CKI␦ Enzymatic Activity. CKI activity was measured
by the IMAP assay using the IMAP Screening Express kit (Molecular Devices),
according to the manufacturer’s protocol. For the P81 phosphocellulose paper

ACKNOWLEDGMENTS. We thank K. Inoue, Y. Takamiya, S. Yoshimura, K. Torii,
N. Esumi, and other staff members of the Animal Resource Unit, RIKEN CDB,
for the care of transgenic animals; H. Omori and Y. Uehara, Research Group for
Super Analyzer Development Technology, ASI, RIKEN, for help with theconstruction of the CCD-Tron system; A. Taya for technical support; J.E. Baggs
for technical information about gene knockdown studies using siRNA; Y.
Koyama for valuable comments; J.B. Hogenesch and E.A. Kuld for valuablecomments and critical reading of the manuscript; C. Johnson and M. Rosbash
for information about the historical work on temperature-compensation andfor suggestions for experiments; and S. Onami, the Developmental Systems
Modeling Team, ASI, RIKEN, for total support. This research was supported by
the National Project on Protein Structural and Functional Analyses (RIKEN),
Grant-in-Aid for Scientific Research on ‘‘Development of Basic Technology toControl Biological Systems Using Chemical Compounds’’ from the New Energy
and Industrial Technology Organization (NEDO) (to H.R.U.), Genome Network
Project grant (to H.R.U.) from the Japanese Ministry of Education, Culture,
Sports, Science and Technology, an intramural Grant-in-Aid from the RIKEN
Center for Developmental Biology (CDB), Director’s Fund from CDB (to H.R.U.),and intramural budget from the Genomic Science Center (to T.K.), and CDB(to H.R.U.), RIKEN; and National Institutes of Health Grant P50 MH074924 (toJ.S.T.). J.S.T. is an Investigator in the Howard Hughes Medical Institute.

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Analysis of the Temperature Sensitivity of mPER2 Degradation in the mPer2Luc
MEFs. The mPer2Luc MEFs cultures were maintained on 24-well culture plate at
27, 32, or 37 °C for 4 days, and the bioluminescence from the cells wasmonitored by the PMT-Tron system until the oscillations of the bioluminescence from mPER2::LUC triggered by the temperature change were diminished. The cells were then treated with 400 ␮g/mL CHX. The bioluminescence
was recorded every 30 min for 5 h by the PMT-Tron system. The time course of
the bioluminescence in each well was normalized and analyzed as described
for the degradation rate of the LUC::mPER2 protein in 293T cells (SI Text).

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